Tight control of gene expression in eucaryotic cells by tetracycline-responsive promoters

ABSTRACT

Transgenic animals carrying two transgenes, the first coding for a transactivator fusion protein comprising a tet repressor and a polypeptide which directly or indirectly activates in eucaryotic cells, and the second comprising a gene operably linked to a minimal promoter operably linked to at least one tet operator sequence, are disclosed. Isolated DNA molecules (e.g., targeting vectors) for integrating a polynucleotide sequence encoding a transactivator of the invention at a predetermined location within a second target DNA molecule by homologous recombination are also disclosed. Transgenic animals having the DNA molecules of the invention integrated at a predetermined location in a chromosome by homologous recombination are also encompassed by the invention. Methods to regulate the expression of a tet operator linked-gene of interest by administering tetracycline or a tetracycline analogue to an animal of the invention are also disclosed. The regulatory system of the invention allows for conditional inactivation or modulation of expression of a gene of interest in a host cell or animal.

RELATED APPLICATIONS

This application is a continuation-in-part of application Ser. No.08/076,327, filed Jun. 14, 1993, now abandoned, the contents of whichare incorporated herein by reference.

BACKGROUND OF THE INVENTION

The study of gene function in complex genetic environments such aseucaryotic cells would greatly profit from systems that would allowstringent control of the expression of individual genes. Ideally, suchsystems would not only mediate an "on/off" status for gene expressionbut would also permit limited expression at a defined level.

Attempts to control gene activity by various inducible eucaryoticpromoters responsive to, for example, heavy metal ions (Mayo et al.,Cell 29:99-108 (1982); Brinster et al., Nature (London) 296:39-42(1982); Searle et al., Nouer, L., CRC Boca Raton, Fla. (1991), pp.167-220), or hormones (Lee et al., Nature (London) 294:228-232 (1981);Hynes et al., Proc. Natl. Acad. Sci. USA 78:2038-2042 (1981); Klock etal., Nature (London) 329:734-736 (1987); Israel & Kaufman, Nucleic AcidsRes. 17:2589-2604 (1989)) have generally suffered from leakiness of theinactive state (e.g., the metallothionein promoter (Mayo et al., Cell29:99-108 (1982)) or from pleiotropic effects caused by the inducingprinciples themselves, such as elevated temperature or glucocorticoidhormone action (Lee et al., Proc. Natl. Acad. Sci, USA 85:1204-1208(1988)).

In search of regulatory systems that do not rely on endogenous controlelements, several groups have demonstrated that the lacrepressor/operator inducer system of Escherichia coli functions ineucaryotic cells. Three approaches have been described: (i) preventionof transcription initiation by properly placed lac operators at promotersites (Hu & Davidson, Cell 48:555-566 (1987); Brown et al., Cell49:603-612 (1987); Figge et al., Cell 52:713-722 (1988); Fuerst et al.,Proc. Natl. Acad. Sci. USA 86:2549-2553 (1989); Deutschle et al., Proc.Natl. Acad. Sci. USA 86:5400-5405 (1989)), (ii) blockage of transcribingRNA polymerase II during elongation by a lac repressor/operator complex(lac R/O; Deutschle et al., Science 248:480-483 (1990)), and (iii)activation of a promoter responsive to a fusion between lacR and theactivating domain of virion protein 16 (VP16) of herpes simplex virus(HSV) (Labow et al., Mol. Cell. Biol. 10:3343-3356 (1990); Baim et al.,Proc. Natl. Acad. Sci. USA 88:5072-5076 (1991)).

At present, however, the utility of the lacR/O-based systems ineucaryotic cells is limited since the inducerisopropyl.β-D-thiogalactopyranoside (IPTG), despite its rapid uptake andintracellular stability (Wyborski & Short, Nucleic Acids Res.19:4647-4653), acts rather slowly and inefficiently, resulting in onlymoderate induction. Nevertheless, an interesting conditional mutant of alacR-VP16 fusion has been described (Baim et al., Proc. Natl. Acad. Sci.USA 88:5072-5076 (1991)). It activates a minimal promoter˜1000-fold atelevated temperatures in the presence of IPTG. The temperaturedependence and the inherent IPTG-related problems, however, may alsolimit this approach.

SUMMARY OF THE INVENTION

This invention features a system for regulating expression of eucaryoticgenes using components of the Tet repressor/operator/inducer system ofprokaryotes. In the system of the invention, transcription of anucleotide sequence operably linked to at least one tet operatorsequence is stimulated by a tetracycline (Tc)-controllabletranscriptional activator fusion protein (referred to herein as tTA).The tTA is comprised of two polypeptides. The first polypeptide is a Tetrepressor (TetR; e.g., a Tn10-derived TetR), which binds to tet operatorsequences in the absence but not the presence of Tc. The secondpolypeptide directly or indirectly activates transcription in eucaryoticcells. For example, the second polypeptide can be a transcriptionalactivation domain from herpes simplex virus virion protein 16 or anothertranscriptional activating domain, e.g. acidic, proline-rich,serine/threonine-rich, glutamine-rich. Alternatively, the secondpolypeptide can be a domain (e.g., a dimerization domain) which recruitsa transcriptional activator (e.g., an endogenous transcriptionalactivator) to interact with the tTA fusion protein by a protein-proteininteraction (e.g., a non-covalent interaction). In the absence of Tc ora Tc analogue, transcription of a gene operably linked to atTA-responsive promoter (typically comprising at least one tet operatorsequence and a minimal promoter) is stimulated by a tTA of theinvention, whereas in the presence of Tc or a Tc analogue, transcriptionof the gene linked to the tTA-responsive promoter is not stimulated bythe tTA.

As described herein, this system functions effectively in transgenicanimals. Accordingly, the invention provides a tetracycline-controllableregulatory system for modulating gene expression in transgenic animals.Additionally, the invention provides targeting vectors for homologousrecombination that enable the components of the regulatory system to beintegrated at a predetermined location in the genome of a host cell oranimal. This embodiment of the invention is able to solve a longstandingproblem in the field generally described as gene targeting or gene knockout. Constitutive disruption of certain genes has been found to producelethal mutations resulting in death of homozygous embryos, e.g., asdescribed for the knock out of the RB gene (Jacks, T. et al. (1992)Nature 359:295-300). This problem precludes the development of "knockout" animals for many genes of interest. The regulatory system of theinvention can be applied to overcome this problem. DNA encoding a tTA ofthe invention can be integrated within a gene of interest such thatexpression of the tTA is controlled by the endogenous regulatoryelements of the gene of interest (e.g., the tTA is expressed spatiallyand temporally in a manner similar to the gene of interest). The gene ofinterest is then operably linked to at least one tet operator sequence(either at its endogenous site by homologous recombination or a secondcopy of the gene of interest, linked to tet operator(s), can beintegrated at another site). Expression of the tet-operator linked geneis thus placed under the control of the tTA, whose pattern of expressionmimics that of the gene of interest. In the absence of Tc, expression ofthe tet operator-linked gene of interest is stimulated by the tTA andthe animal develops like a nonmutated wildtype animal. Then, at aparticular stage of development, expression of the gene of interest canbe switched off by raising the level of Tc (or a Tc analogue) in thecirculation and the tissues of the animal by feeding or injecting Tc (ora Tc analogue) to the animal, thereby inhibiting the activity of the tTAand transcription of the gene of interest. This method is generallyreferred to herein as a "conditional knockout".

Accordingly, one aspect of the invention relates to targeting vectorsfor homologous recombination. In one embodiment, the invention providesan isolated DNA molecule for integrating a polynucleotide sequenceencoding a tetracycline-controllable transactivator (tTA) at apredetermined location in a second target DNA molecule. In this DNAmolecule, a polynucleotide sequence encoding a tTA is flanked at 5' and3' ends by additional polynucleotide sequences of sufficient length forhomologous recombination between the DNA molecule and the second targetDNA molecule at a predetermined location. Typically, the target DNAmolecule into which the tTA-coding sequences are integrated is a gene ofinterest, or regulatory region thereof, in a eucaryotic chromosome in ahost cell. For example, tTA-coding sequences can be inserted into a genewithin a yeast, fungal, insect or mammalian cell. Additionally,tTA-coding sequences can be inserted into a viral gene present within ahost cell, e.g. a baculovirus gene present in insect host cell. In apreferred embodiment, integration of the tTA-encoding sequences into apredetermined location in a gene of interest (by homologousrecombination) places the tTA-coding sequences under the control ofregulatory elements of the gene of interest (e.g., 5' flankingregulatory elements), such that the tTA is expressed in a spatial andtemporal manner similar to the gene of interest.

In another embodiment of the targeting vector for homologousrecombination, the isolated DNA molecule permits integration of apolynucleotide sequence encoding both a tTA and a tTA-responsivepromoter within a predetermined gene of interest in a second target DNAmolecule (a "single hit vector", schematically illustrated in FIGS.13A-B). This molecule includes: 1) a first polynucleotide sequencecomprising a 5' flanking regulatory region of the gene of interest,operably linked to 2) a second polynucleotide sequence encoding a tTA;and 3) a third polynucleotide sequence comprising a tTA-responsivepromoter, operably linked to: 4) a fourth polynucleotide sequencecomprising at least a portion of a coding region of the gene ofinterest. The first and fourth polynucleotide sequences are ofsufficient length for homologous recombination between the DNA moleculeand the gene of interest such that expression of the tTA is controlledby 5' regulatory elements of the gene of interest and expression of thegene of interest is controlled by the tTA-responsive promoter (i.e.,upon binding of the tTA to the tTA-responsive promoter, expression ofthe gene of interest is stimulated). This targeting vector can alsoinclude a polynucleotide sequence encoding a selectable marker operablylinked to a regulatory sequence. Typically, the selectable markerexpression unit is located between the tTA-encoding sequence (i.e., thesecond polynucleotide sequence described above) and the tTA-responsivepromoter (i.e., the third polynucleotide sequence described above).Additionally or alternatively, this targeting vector can also include asequence, typically located upstream (i.e., 5') of the tTA-responsivepromoter (e.g., between the selectable marker expression unit and thetTA responsive promoter) which terminates transcription or otherwiseinsulated downstream elements from the effects of upstream regulatoryelements. The tTA-responsive promoter typically includes a minimalpromoter operably linked to at least one tet operator sequence. Theminimal promoter is derived, for example, from a cytomegalovirusimmediate early gene promoter or a herpes simplex virus thymidine kinasegene promoter.

Another aspect of the invention relates to eucaryotic host cellscontaining a DNA molecule encoding a tTA integrated at a predeterminedlocation in a second target DNA molecule (e.g., a gene of interest) inthe host cell. Such a eucaryotic host cell can be created by introducinga targeting vector of the invention into a population of cells underconditions suitable for homologous recombination between the DNAencoding the tTA and the second target DNA molecule and selecting a cellin which the DNA encoding the tTA has integrated at a predeterminedlocation within the second target DNA molecule. The host cell can be amammalian cell (e.g., a human cell). Alternatively, the host cell can bea yeast, fungal or insect cell (e.g., the tTA-encoding DNA can beintegrated into a baculovirus gene within an insect cell). A preferredhost cell type for homologous recombination is an embryonic stem cell,which can then be used to create a non-human animal carrying tTA-codingsequences integrated at a predetermined location in a chromosome of theanimal. A host cell can further contain a gene of interest operablylinked to a tTA-responsive transcriptional promoter. The gene ofinterest operably linked to the tTA-responsive promoter can beintegrated into DNA of the host cell either randomly (e.g., byintroduction of an exogenous gene) or at a predetermined location (e.g.,by targeting an endogenous gene for homologous recombination). The genelinked to the tTA-responsive promoter can be introduced into the hostcell independently from the DNA encoding the tTA, or alternatively, a"single hit" targeting vector of the invention can be used to integrateboth tTA-coding sequences and a tTA-responsive promoter into apredetermined location in DNA of the host cell. Expression of a gene ofinterest operably linked to a tTA-responsive promoter in a host cell ofthe invention can be inhibited by contacting the cell with tetracyclineor a tetracycline analogue.

Another aspect of the invention relates to non-human transgenic animalshaving a transgene comprising a polynucleotide sequence encoding atetracycline-controllable transactivator (tTA) of the invention orhaving a transgene encoding a gene of interest operably linked to atTA-responsive promoter. Double transgenic animals having bothtransgenes (i.e., a tTA-coding transgene and a gene of interest linkedto a tTA-responsive promoter) are also encompassed by the invention. Inone embodiment, the transgenic animal is a mouse. In other embodiments,the transgenic animal is a cow, a goat, a sheep and a pig. Transgenicanimals of the invention can be made, for example, by introducing a DNAmolecule encoding the tTA or the gene of interest operably linked to atTA responsive promoter into a fertilized oocyte, implanting thefertilized oocyte in a pseudopregnant foster mother, and allowing thefertilized oocyte to develop into the non-human transgenic animal tothereby produce the non-human transgenic animal. Double transgenicanimals can be created by appropriate mating of single transgenicanimals. Expression of a gene of interest operably linked to a tTAresponsive promoter in a double transgenic animal of the invention canbe inhibited by administering tetracycline or a tetracycline analogue tothe animal.

Another aspect of the invention relates to non-human transgenic animalshaving a transgene encoding a tTA of the invention, wherein thetransgene is integrated by homologous recombination at a predeterminedlocation within a chromosome within cells of the animal (also referredto herein as a homologous recombinant animal). The homologousrecombinant animal can also have a second transgene encoding a gene ofinterest operably linked to a tTA-responsive promoter. The secondtransgene can be introduced randomly or, alternatively, at apredetermined location within a chromosome (e.g., by homologousrecombination. For example, a single hit vector of the invention can beused to create a homologous recombinant animal in which expression ofthe tTA is controlled by 5' regulatory elements of a gene of interestand expression of the gene of interest is controlled by thetTA-responsive promoter (such that in the absence of Tc, expression ofthe gene is stimulated by tTA binding to the tTA responsive promoter).

A non-human transgenic animal of the invention having tTA-codingsequences integrated at a predetermined location within chromosomal DNAof cells of the animal can be created by introducing a targeting vectorof the invention into a population of embryonic stem cells underconditions suitable for homologous recombination between the DNAencoding the tTA and chromosomal DNA within the cell, selecting anembryonic stem cell in which DNA encoding the tTA has integrated at apredetermined location within the chromosomal DNA of the cell,implanting the embryonic stem cell into a blastocyst, implanting theblastocyst into a pseudopregnant foster mother and allowing theblastocyst to develop into the non-human transgenic animal to therebyproduce the non-human transgenic animal.

Another aspect of the invention relates to a process for producing andisolating a gene product (e.g., protein) encoded by a gene of interestoperably linked to a tTA-responsive transcriptional promoter in a hostcell of the invention carrying tTA-coding sequences. In the process, ahost cell is first grown in a culture medium in the presence oftetracycline or a tetracycline analogue (under these conditions,expression of the gene of interest is not stimulated). Next, theconcentration of tetracycline or the tetracycline analogue in theculture medium is reduced to stimulate transcription of the gene ofinterest. The cells are further cultured until a desired amount of thegene product encoded by the gene of interest is produced by the cells.Finally, the gene product is isolated from harvested cells or from theculture medium. Preferred cells for use in the process include yeast orfungal cells.

Kits containing the components of the regulatory system of the inventiondescribed herein are also within the scope of the invention.

Various additional features, components and aspects of this inventionare described in further detail below.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1a-1b: Schematic representation of tetR-VP 16 fusion proteins(tTAs), encoded by plasmids pUHD15-1 and pUHD151-1, and a tTA-dependenttranscription unit, encoded by plasmid pUHC13-3.

FIG. 1a: Diagrammatic representation of two tTA proteins. In both fusionproteins, tTA and tTA_(S), the original 207-amino-acid sequence of tetRis conserved. Two versions of VP 16 sequences encoding the activationdomain were fused in frame to the 3' end of the tetR gene, resulting intTA and tTAs. The bold letters indicate the original amino acids at theN terminal end, the junction, and the C-terminal end of the fusionproteins; the other letters designate amino acids introduced due tosequence constraints of the particular system. The numbers delineateamino acid positions within tetR (Hillen and Wissman in Protein-NucleicAcid Interaction, Topics in Molecular and Structural Biology, Saengerand Heinemann (eds.), Vol 10, pp. 143-162 (1989)) or VP16 (Treizenberget al., Genes Dev. 2:718-729 (1988)), respectively.

FIG. 1b: The tTA-dependent transcriptional unit consists of the simianvirus 40 (SV40) poly(A) site (An), the luciferase gene (luc), theP_(hCMV) *-1 or P_(hCMV) *-2. The two promoters encompass the sequencebetween +75 and -53 of the P_(hCMV) *-2 with one base-pair exchange at-31, which creates a Stu I cleavage site. The Xho I site introduced at-53 by PCR was utilized to insert the heptamerized tetO sequence. Thisheptameric sequence is flanked at one side by an 18-nucleotidepolylinker, which allows the insertion of the operators in bothorientations as Sal l/Xhol fragments. The position of the central G/Cbase pair of the promoter proximal operator to position +1 is -95 forP_(hCMV) *-I (upper construct) and -76 for P_(hCMV) *-2 (lowerconstruct). The plasmids that contain the four constructs are indicatedon the far right.

FIGS. 2a-2b. Western blots showing the identification andcharacterization of tTA produced in HeLa cells. HeLa cells grown to 40%confluency were transiently transfected with pUHD15-1 by the calciumphosphate method. Nuclear and cytoplasmic extracts were prepared after36 hr.

FIG. 2a: Western blot analysis of electrophoretically separated extracts(6% acrylamide/0.1% SDS gels) with tetR-specific antibodies reveals aprotein of about 37 kDa (tTA) in cytoplasmic (C) and nuclear (N)extracts in pUHD15-1 transfected cells (+) that is not present inmock-transfected cells (-).

FIG. 2b: Mobility change of tetO DNA by tTA binding from HeLa cellnuclear extracts. Radioactively labeled tetO DNA was mixed with extractsfrom mock-transfected (lanes 2 and 3) and pUHD15-1-transfected (lanes 4and 5) HeLa cells in the absence (lanes 2 and 4) and presence (lanes 3and 5) of 1 μg of tetracycline per ml (added 2 min prior to the additionof the operator). Lane 1 contains labeled operator DNA only.

FIGS. 3a-3b. Graphs showing the dependence of tTA function ontetracycline.

FIG. 3a: Dependence of luciferase (luc.) activity on tetracyclineconcentration. HeLa cell clones X1 (dashed line) and T12 previouslygrown in tetracycline-free medium were seeded with a density of 5000cells per 35 mm dish and incubated at the tetracycline concentrationsindicated. After reaching ˜90% confluency, cells were harvested andassayed for luciferase activity. Data given are the means ±SD of threeindependent experiments.

FIG. 3b: Kinetics of tetracycline action. X1 cells were grown in 100 mmdishes to ˜80% confluency in the absence or presence (0.1 μg/ml) oftetracycline. At time 0, cells were washed with phosphate-bufferedsaline and split into smaller culture dishes (1/20th of the initialcultures per 35 mm dish). Half of the cultures remained intetracycline-free medium (▪) and the other half were incubated in thepresence of tetracycline (1 μg/ml; □). The X1 culture grown intetracycline-containing medium was split in the same manner; one halfwas further incubated in the presence of tetracycline (), whereas theother half was transferred to tetracycline-free medium (◯). At the timesindicated, aliquots were harvested and examined for luciferase activity.The slight increase in luciferase activity monitored at 4 hr in theculture containing tetracycline () is reproducible and reflectsluciferase induction during the washing step.

FIGS. 4a-4d. [SEQ ID NO: 1] The polynucleotide sequence coding for tTAtransactivator.

FIGS. 5a-5d. [SEQ ID NO: 3] The polynucleotide sequence coding fortTA_(S) transactivator.

FIG. 6. [SEQ ID NO: 5] The polynucleotide sequence of P_(hCMV) *-1. Thenucleotide sequence shown encompasses the tet operator sequences(italics) and the hCMV minimal promoter, of which position -53, the TATAbox and position +75 (relative to the transcription start site) areunderlined.

FIG. 7. [SEQ ID NO: 6] The polynucleotide sequence of P_(hCMV) *-2. Thenucleotide sequence shown encompasses the tet operator sequences(italics) and the hCMV minimal promoter, of which position -53, the TATAbox and position +75 (relative to the transcription start site) areunderlined.

FIG. 8 [SEQ ID NO: 7] The polynucleotide sequence of PTk*-1. Thenucleotide sequence shown encompasses the tet operator sequences(italics) and the HSV-Tk minimal promoter, of which position -81, theTATA box and position +7 (relative to the transcription start site) areunderlined.

FIGS. 9a-9h. [SEQ ID NO: 8] The polynucleotide sequence of the cDNAcoding for the rabbit progesterone receptor under control of P_(hCMV)*-1.

FIGS. 10a-10f. [SEQ ID NO: 9] The polynucleotide sequence of the cDNAcoding for the rabbit progesterone receptor under control of P_(hCMV)*-1.

FIG. 11. A schematic representation of Conditional Knock Out Strategy 1in which "E.G. 5'" represents flanking nucleotide sequence from 5' ofthe coding sequence for an Endogenous Gene; "E.G. 3'" representsflanking nucleotide sequence from 3' of the coding sequence for anEndogenous Gene; and "tTARE" represents a tTA responsive elementinserted just upstream of a copy of the endogenous gene of interest. (Inother embodiments the gene linked to the tTA is a heterologous gene.)

FIG. 12. A schematic representation of Conditional Knock Out Strategy 2in which "tTARE" is a tTA responsive promoter element: "E.G". is anendogenous gene; "E.G. 5'" represents flanking nucleotide sequence from5' of the coding sequence for an Endogenous Gene; "E.G. 3'" representsflanking nucleotide sequence from 3' of the coding sequence for anEndogenous Gene; and "TK" is a thymidine kinase gene.

FIGS. 13a-13b A schematic representation of Conditional Knock OutStrategy 3 depicting vector designs in which abbreviations are asdefined above, Neo^(r) is a neomycin resistance gene and pPGK isphosphoglycerate kinase sequence.

FIG. 14. A graphic representation of the doxycycline dependentluciferase activity in double transgenic mice carrying P_(hCMV) -tTA andP_(hCMV) *-1-luc transgenes. Light bars show tTA-activated luciferaselevels in different tissues from 2 individual mice. Dark bars showluciferase levels in different tissues from 2 individual mice thatreceived doxycycline in the drinking water (200 mg/ml, 5% sucrose) for 7days. Spotted bars (controls) represent the average luciferasebackground activity from 5 individuals from line L7, carrying only theP_(hCMV) *-1-luc transgene.

DETAILED DESCRIPTION OF THE INVENTION DEFINITIONS

The description that follows makes use of a number of terms used inrecombinant DNA technology. In order to provide a clear and consistentunderstanding of the specification and claims, including the scope to begiven such terms, the following definitions are provided.

Cloning Vector.

A plasmid or phage DNA or other DNA sequence which is able to replicateautonomously in a host cell, and which is characterized by one or asmall number of restriction endonuclease recognition sites at which suchDNA sequences may be cut in a determinable fashion without loss of anessential biological function of the vector, and into which a DNAfragment may be spliced in order to bring about its replication andcloning. The cloning vector may further contain a marker suitable foruse in the identification of cells transformed with the cloning vector.

Expression Vector.

A vector similar to a cloning vector but which is capable of enhancingthe expression of a gene which has been cloned into it, aftertransformation into a host. The cloned gene is usually placed under thecontrol of (i.e., operably linked to) certain control sequences such aspromoter sequences. Promoter sequences may be either constitutive orinducible.

Eucaryotic Host Cell.

According to the invention, a eucaryotic host cell may be any such cellwhich include, but are not limited to, yeast cells, plant cells, fungalcells, insect cells, e.g. Schneider and sF9 cells, mammalian cells, e.g.HeLa cells (human), NIH3T3 (murine), RK13 (rabbit) cells, embryonic stemcell lines, e.g, D3 and J1, and cell types such as hematopoietic stemcells, myoblasts, hepatocytes, lymphocytes, airway epithelium and skinepithelium.

Recombinant Eucaryotic Host.

According to the invention, a recombinant eucaryotic host may be anyeucaryotic cell which contains the polynucleotide molecules of thepresent invention on an expression vector or cloning vector. This termis also meant to include those eucaryotic cells that have beengenetically engineered to contain the desired polynucleotide moleculesin the chromosome, genome or episome of that organism. Thus, therecombinant eucaryotic host cells are capable of stably or transientlyexpressing the proteins.

Recombinant vector.

Any cloning vector or expression vector which contains thepolynucleotide molecules of the invention.

Host.

Any prokaryotic or eucaryotic cell that is the recipient of a replicablevector. A "host" as the term is used herein, also includes prokaryoticor eucaryotic cells that can be genetically engineered by well knowntechniques to contain desired gene(s) on its chromosome or genome. Forexamples of such hosts, see Sambrook et al., Molecular Cloning: ALaboratory Manual, Second Edition, Cold Spring Harbor Laboratory, ColdSpring Harbor, N.Y. (1989).

Promoter.

A DNA sequence generally described as the 5' region of a gene, locatedproximal to the start codon. The transcription of an adjacent gene(s) isinitiated at the promoter region. If a promoter is an induciblepromoter, then the rate of transcription increases in response to aninducing agent. In contrast, the rate of transcription is not regulatedby an inducing agent if the promoter is a constitutive promoter.

Minimal Promoter.

A partial promoter sequence which defines the transcription start sitebut which by itself is not capable, if at all, of initiatingtranscription efficiently. The activity of such minimal promotors dependon the binding of activators such as a tetracycline-controlledtransactivator to operably linked binding sites.

Gene.

A DNA sequence that contains information needed for expressing apolypeptide or protein.

Structural Gene.

A DNA sequence that is transcribed into messenger RNA (mRNA) that isthen translated into a sequence of amino acids characteristic of aspecific polypeptide.

Polynucleotide molecules.

A polynucleotide molecule may be a polydeoxyribonucleic acid molecule(DNA) or a polyribonucleic acid molecule (RNA).

Complementary. DNA (cDNA).

A "complementary DNA" or "cDNA" gene includes recombinant genessynthesized by reverse transcription of mRNA and from which interveningsequences (introns) have been removed.

Expression.

"Expression" is the process by which a polypeptide is produced from astructural gene. The process involves transcription of the gene intomRNA and the translation of such mRNA into polypeptide(s).

Fragment.

A "fragment` of a molecule is meant to refer to any polypeptide subsetof that molecule.

Tet repressor.

A "tet repressor" refers to a prokaryotic protein which binds to a tetoperator sequence in the absence but not the presence of tetracycline.The term "tet repressor" is intended to include repressors of differentclass types, e.g., class A, B, C, D or E tet repressors.

Tetracycline Analogue.

A "tetracycline analogue" is any one of a number of compounds that areclosely related to tetracycline (Tc) and which bind to the tet repressorwith a Ka of at least about 10⁶ M⁻¹. Preferably, the tetracyclineanalogue binds with an affinity of about 10⁹ M⁻¹ or greater, e.g. 10⁹M⁻¹. Examples of such tetracycline analogues include, but are notlimited to those disclosed by Hlavka and Boothe, "The Tetracyclines," inHandbook of Experimental Pharmacology 78, R. K. Blackwood et al. (eds.),Springer Verlag, Berlin-New York, 1985; L. A. Mitscher "The Chemistry ofthe Tetracycline Antibiotics, Medicinal Research 9, Dekker, New York,1978; Noyee Development Corporation, "Tetracycline ManufacturingProcesses," Chemical Process Reviews, Park Ridge, N.J., 2 volumes, 1969;R. C. Evans, "The Technology of the Tetracyclines," BiochemicalReference Series 1, Quadrangle Press, New York, 1968; and H. F. Dowling,"Tetracycline," Antibiotics Monographs, no. 3, Medical Encyclopedia, NewYork, 1955; the contents of each of which are fully incorporated byreference herein. Examples of tetracycline analogues includeanhydrotetracycline, doxycycline, chlorotetracycline,epioxytetracycline, and the like. Certain Tc analogues, such asanhydrotetracycline and epioxytetracycline, have reduced antibioticactivity compared to Tc.

Transgenic Animal.

A transgenic animal is an animal having cells that contain a transgene,wherein the transgene was introduced into the animal or an ancestor ofthe animal at a prenatal, e.g., an embryonic, stage. A transgene is aDNA which is integrated into the genome of a cell from which atransgenic animal develops and which remains in the genome of the matureanimal, thereby directing the expression of an encoded gene product inone or more cell types or tissues of the transgenic animal. Non-humananimals into which transgenes can be introduced by techniques known inthe art include mice, goats, sheep, pigs, cows and other domestic farmanimals.

A transgenic animal can be created, for example, by introducing anucleic acid encoding a protein of interest (typically linked toappropriate regulatory elements, such as a constitutive ortissue-specific enhancer) into the male pronuclei of a fertilizedoocyte, e.g., by microinjection, and allowing the oocyte to develop in apseudopregnant female foster animal. Intronic sequences andpolyadenylation signals can also be included in the transgene toincrease the efficiency of expression of the transgene. Methods forgenerating transgenic animals, particularly animals such as mice, havebecome conventional in the art and are described, for example, in U.S.Pat. Nos. 4,736,866 and 4,870,009 and Hogan, B. et al., (1986) ALaboratory Manual, Cold Spring Harbor, N.Y., Cold Spring HarborLaboratory. A transgenic founder animal can be used to breed additionalanimals carrying the transgene. A transgenic animal carrying onetransgene can further be bred to another transgenic animal carrying asecond transgenes to create a so-called "double transgenic" animalcarrying two transgenes.

Homologous Recombinant Animal.

The term "homologous recombinant animal" as used herein is intended todescribe an animal containing a gene which has been modified byhomologous recombination between the gene and a DNA molecule introducedinto an embryonic cell of the animal, or ancestor thereof. Thus, ahomologous recombinant animal is a type of transgenic animal in whichthe transgene is introduced into a predetermined chromosomal location inthe genome of the animal by homologous recombination.

To create such a homologous recombinant animal, a vector is preparedwhich contains DNA of interest (e.g., encoding a tTA of the invention)flanked at its 5' and 3' ends by additional nucleic acid of a eucaryoticgene of interest at which homologous recombination is to occur. Theflanking nucleic acid is of sufficient length for successful homologousrecombination with the eucaryotic gene. Typically, several kilobases offlanking DNA (both at the 5' and 3' ends) are included in the vector(see e.g., Thomas, K. R. and Capecchi, M. R. (1987) Cell 51:503 for adescription of homologous recombination vectors). The vector isintroduced into an embryonic stem cell line (e.g., by electroporation)and cells in which the introduced DNA has homologously recombined withthe endogenous DNA are selected (see e.g., Li, E. et al. (1992) Cell69:915). The selected cells are then injected into a blastocyst of ananimal (e.g., a mouse) to form aggregation chimeras (see e.g., Bradley,A. in Teratocarcinomas and Embryonic Stem Cells: A Practical Approach,E. J. Robertson, ed. (IRL, Oxford, 1987) pp. 113-152). A chimeric embryocan then be implanted into a suitable pseudopregnant female fosteranimal and the embryo brought to term. Progeny harbouring thehomologously recombined DNA in their germ cells can be used to breedanimals in which all cells of the animal contain the homologouslyrecombined DNA by so-called "germline transmission". Animals carryingthe recombined gene can be bred to homozygosity and/or bred with otheranimals carrying other transgenes.

Recombinant expression of proteins is commonly done using constitutivepromoters like human CMV (Boshart, M. et al. 1985, Cell Vol. 41,521-530) or the adenovirus major late promoter or SV40 early promoter asdescribed below (see also, e.g., Kaufman, R. J. 1990 Meth Enzymol. Vol.185:537-566 and Benoist C. et al. (1981) Nature Vol.290:304 ff).However, in the case of proteins such as certain proteases, cytotoxic orcytostatic proteins that interfere with the cell membranes or proteinslike certain receptors, whose normal biological function triggers aresponse to the host cell environment (media components, temperatureetc.) that is detrimental to the host cell, expression of the proteinsmay negatively effect the physiology of the host cell. In other casesoverexpression of a desired gene may simply be unduly taxing for theproducing cells. In such cases it is desirable to inhibit the expressionof the desired gene until an optimal cell density has been achieved, andonly then, after an optimal period of cell culture in vitro or cellgrowth and development in vivo (determined empirically), induce geneexpression in the cells to produce sufficient quantities of the protein.While a number of systems have been proposed and tried (as generallyreviewed by Yarranton, G. T. 1992 Current Opinion in Biotechnology Vol.3:506-511) many such systems do not allow for tight repression andsubsequent complete activation. Others employ impractical activationsteps that are not expected to be useful in large scale fermentation orin whole animals. The current invention however fulfills all thesecriteria in eucaryotic expression systems using a transcriptional switchbased on procaryotic control elements.

Aspects of the tightly regulatable genetic switch used in this inventionfor controlling gene transcription are described in Gossen & Bujard,1992, Proc. Natl. Acad. Sci. USA 89:55475551 and in U.S. patentapplication Ser. No. 08/076,726, entitled "Tight Control of GeneExpression in Eucaryotic Cells by Tetracycline-responsive Promoters"filed 14 Jun. 1993, the full contents of both of which are incorporatedherein by reference.

The genetic switch employed in this invention comprises two components:(i) a polynucleotide (e.g. DNA) moiety encoding atetracycline-controllable transcriptional activator (also referred toherein as a "transactivator" or tTA) and (ii) a gene of interestoperably linked to, i.e., under the transcriptional control of, apromoter responsive to the transcriptional activator.

The tetracycline-controllable transactivator (tTA) is composed of aprocaryotic tet repressor (tetR) (also referred to as the firstpolypeptide) operably linked to a polypeptide which directly orindirectly activates transcription in eucaryotic cells (also referred toas the second polypeptide). Typically, nucleotide sequences encoding thefirst and second polypeptides are ligated to each other in-frame tocreate a chimeric gene encoding a fusion protein, although the first andsecond polypeptides can be operably linked by other means that preservethe function of each polypeptide (e.g., chemically crosslinked). In oneembodiment, the second polypeptide is a transcriptional activatingprotein such as the acidic transactivating domain of virion protein 16(VP16) of herpes simplex virus (HSV) as in plasmids pUHD15-1 orpUHD151-1 (see FIG. 11). It should be appreciated that othertransactivators, including acidic, proline- or serine/threonine- orglutamine-rich transactivating moieties as described below, may besubstituted for the VP16 transactivator in the tetracycline-controllablefusion transactivator. In this embodiment, the second polypeptide of thefusion protein is capable of directly activating transcription.

In another embodiment, the second polypeptide of the tTA fusion proteinindirectly activates transcription by recruiting a transcriptionalactivator to interact with the tetR fusion protein. For example, tetRcan be fused to a polypeptide domain (e.g., a dimerization domain)capable of mediating a protein-protein with a transcriptional activatorprotein, such as an endogenous activator present in a host cell. It hasbeen demonstrated that functional associations between DNA bindingdomains and transactivation domains need not be covalent (see e.g.,Fields and Song (1989) Nature 340:245-247; Chien et al. (1991) Proc.Natl. Acad. Sci. USA 88:9578-9582; Gyuris et al. (1993) Cell 75:791-803;and Zervos, A. S. (1993) Cell 72:223-232). Accordingly, the secondpolypeptide of the tTA fusion protein may not directly activatetranscription but rather may form a stable interaction with anendogenous polypeptide beating a compatible protein-protein interactiondomain and transactivation domain. Examples of suitable interaction (ordimerization) domains include leucine zippers (Landschulz et al. (1989)Science 243:1681-1688), helix-loop-helix domains (Murre, C. et al.(1989) Cell 58:537-544) and zinc finger domains (Frankel, A. D. et al.(1988) Science 240:70-73). Interaction of a dimerization domain presentin the tTA fusion protein with an endogenous nuclear factor results inrecruitment of the transactivation domain of the nuclear factor to thetTA, and thereby to a tet operator sequence to which the tTA is bound.

A variation of this approach is to construct a fusion of the tetR DNAbinding sequence to the non-DNA binding amino acid sequences of the TATAbinding protein (TBP) (TBP is described in Kao, C. C. et al. (1990)Science 248:1646-1650). The DNA binding form of TBP is part of a proteincomplex designated TFIID. The function of TBP in the complex is torecruit other protein components of the TFIID complex to position nearthe transcription initiation site of eucaryotic genes containing a TATAbox (i.e., TBP binding site). When bound to the TATA box, the TFIIDcomplex subsequently mediates the sequential recruitment of othermembers of the basic transcriptional initiation complex, resulting ininitiation of transcription (described in more detail in Buratowski, S.et al. (1989) Cell 56:549-561). Accordingly, when fused to tetR DNAbinding sequences, TBP may recruit other members of the basictranscription initiation complex to DNA sequences containing a tetoperator(s). Furthermore, by substituting a TATA sequence present in aeukaryotic gene of interest with a tet operator(s), the tetR/TBP fusionprotein can be targeted to this site in a manner dependent on thepresence or absence of Tc (or analogue thereof), resulting inTc-dependent initiation of transcription. Since, in this approach, thegene of interest to be regulated by the tTA (i.e., tetR/TBP fusionprotein) lacks a functional TATA element, the basal level of expressionof the gene in the presence of Tc (or analogue) is expected to be verylow. However, upon removal of Tc (or analogue), transcription initiationis restored via binding of tetR/TBP to the tet operator(s) andrecruitment of other components of the transcription initiation complex.

The tTA may be expressed in a desired host cell using otherwiseconventional methods and materials by transfecting or transforming thehost cell with the tTA-encoding DNA operably linked to a conventionalpromoter (such as are mentioned elsewhere herein), e.g. for constitutiveexpression.

The second component of the genetic switch is the tTA-responsivetranscriptional promoter to which the gene of interest is operablylinked. The promoter may be a minimal promoter comprising, for example,a portion of the cytomegalovirus (CMV) IE promoter, operably linked toat least one tet operator sequence, derived for example from thetetracycline resistance operon encoded in Tn10 of E. coli (Hillen &Wissmann, "Topics in Molecular and Structural biology" inProtein-Nucleic Acid Interaction, Saeger & Heinemannn eds., Macmillan,London, 1989, Vol.10, pp.143-162), to serve as target sequences for atTA.

Other suitable minimal promoters include PhCMV*-1, PhCMV*-2, and PtK*-1,described herein, or other minimal promoters derived from promoterelements typically used in the cell line employed as described in thereferences throughout this application.

Minimal promoter elements particularly useful for a given cell line maybe selected from a series of deletion mutants of the original promoternucleotide sequence, based on the ability of a given member of theseries (for instance, placed as a Xhol/Sacll fragment into thecorresponding restriction sites of plasmid pUHC13-3) to be activated intransient transfection experiments using a cell line stably expressingthe tetR-VP16 fusion protein; as will be appreciated a cell line stablyexpressing any other fusion of tetR with a protein domain capable ofactivating transcription (see below) can be used. As will also beappreciated plasmid pUHC13-3 may be modified for the specificapplication by replacing genetic elements like poly-adenylation sites orsplice sites with those functioning in the cell line in question.Specific details may be found in the references throughout thisapplication or references cited therein, the full contents of which areincorporated herein by reference. A second criterion for the selectionof the optimal minimal promoter is the degree of repression in thepresence of tetracycline (see below). Typically the deletion mutant withthe highest activation factor as described below is chosen.

Promoter deletion mutants may be prepared as generally described byRosen, C. et al (1985) Cell Vol. 41, 813-823 or Nelson C. et al. (1986)Nature Vol. 322, 557-562. Other methods, including methods useful in thepreparation of stable tetR-VP16 cell lines, are essentially as describedin "Current Protocols in Molecular Biology" Ausubel, F. M. et al (eds.)1989 Vol. 1 and 2 and all supplements to date Greene PublishingAssociates and Wiley-Interscience, John Wiley & Sons, New York, the fullcontents of which are incorporated herein by reference, or as describedin the other references cited throughout this application.

The presence of tet operator element(s) renders such recombinantpromoter moieties responsive to the tTA of the invention. In HeLa cellsconstitutively expressing the TetR-VP16 tTA, high levels of luciferaseexpression have been achieved under the control of such a modified CMVpromoter sequence. The incorporation of the tetR domain within the tTArenders this expression system sensitive to the presence oftetracycline. The binding of tetracycline to the tetR domain of the tTAprevents the tTA from exerting its transactivating effects. Depending onthe concentration of tetracycline in the culture medium (0-1 μg/ml), theluciferase activity can be regulated up to five orders of magnitude inthe previously mentioned example. This system provides both a reversibleon/off switch and a differential control--as desired--for regulatinggene expression in eucaryotic hosts. It should be appreciated thattetracycline analogs which are capable of specific functionalinteraction with tetR may be used in place of tetracycline,

A eucaryotic production cell line of this invention is preparedaccording to the design described above. Assembly of the components andincorporation thereof into a eucaryotic host cell are conducted byotherwise conventional methods such as are described generally byKriegler, M. (editor), 1990, Gene Transfer and Expression, A LaboratoryManual (Stockton Press). Care should to be taken to select forintegration of the gene of interest into a chromosomal site thatexhibits sufficiently low basal expression when, or to the extent,desired (see e.g. Table 1). The recombinant host cell obtained is grownin the presence of tetracycline or tetracycline analogues until anoptimal density that has been determined empirically to allow forsubsequent induction of gene expression. After the desired cell densityhas been reached gene expression is induced by dilution and/or removalof the tetracycline or analog thereof. The culture is then continuouslygrown until a optimal expression level has been reached. The recombinantprotein is then harvested according to standard procedures.

The use of eucaryotic cells as host cells for expression of recombinantproteins is generally reviewed in M. Kriegler 1990 "Gene Transfer andExpression, A Laboratory Manual". Stockton Press., incorporated hereinas reference. While CHO^(dhfr-) cells (Urlaub, G. and Chasin (1980)Proc. Natl. Acad. Sci. USA 77:4216-4220), 293 cells (Graham, F. L. etal. (1977) J. Gen. Virol. 36: pp. 59) or myeloma cells like SP2 or NSO(Galfre, G. and Milstein, C. (1981) Meth Enzymol. 73 (B):3-46) arecommonly used it should be clear to those of ordinary skill in the art,that any eucaryotic cell line can be used in the practice of the subjectinvention, so long as the cell line is not incompatible with the proteinto be expressed, the selection system chosen or the fermentation systememployed. This invention is broadly applicable and encompassesnon-mammalian eucaryotic cells as well, including insect (e.g. Sp.frugiperda), yeast (e.g. S. cerevisiae, S. pombe, H. polymorpha) andfungal cells, containing and capable of expressing the two components ofthe foregoing genetic switch.

The eucaryotic host cells used for regulated expression in thisinvention may thus be yeast cells including, but not limited toSaccharomyces cerevisiae, Pichia pastoris, Kluyveromyces lactis andHansenula polymorpha, as generally reviewed by Fleer, R. (1992), CurrentOpinion in Biotechnology Vol. 3, No. 5: p. 486-496, the full contentsthereof and of which the references cited therein are incorporatedherein by reference.

In other embodiments the eucaryotic cells used for regulated expressionare insect cells carrying in their chromosomes the heterologous DNAmoiety encoding a transactivator fusion protein (tTA) comprising atetracycline repressor and a protein capable of activating transcriptionin the host cell. A second recombinant DNA moiety encoding the gene ofinterest operably linked to a promoter responsive to the transcriptionalactivator is carried on the baculovirus genome. Suitable general methodswhich may be used in the practice of these aspects of the invention arereviewed by O'Reilly et al. (1992) "Baculovirus expression vectors, ALaboratory Manual" Stockton Press, the full contents of which areincorporated herein by reference.

While the gene of interest may be a heterologous gene, i.e. nototherwise present in the parental host cell genome, an important aspectof this invention relates to the regulation of an endogenous gene ofinterest. In such cases the host cell is genetically engineered toinsert into the host cell genome the tTA-responsive promoter such thatthe desired endogenous gene is under the transcriptional control of thetTA-responsive promoter. This may be accomplished for example by linkinga copy of the endogenous gene to the tTA-responsive promoter andtransfecting or transforming the host cell with the recombinantconstruct. In one approach, the construct is introduced by homologousrecombination into the loci of the endogenous gene. Briefly, the tTAresponsive promoter is flanked on the 5' side by sufficient DNAsequences from the upstream region but excluding the actual promoterregion of the endogenous gene and on the 3' end by sequencesrepresenting the coding region of the endogenous gene. The extent of DNAsequence homology necessary for homologous recombination is discussedbelow.

In other approaches that construct is inserted at another genetic locus,either predetermined or at random. In any case, the eucaryotic cell isalso transformed or transfected with the DNA construct permittingexpression of the tTA. Alternatively, the DNA construct encoding the tTAmay itself be inserted at the locus of the endogenous gene of interestand the DNA moiety encoding the gene of interest operably linked to atTA-responsive promoter may be introduced elsewhere in the genome. Inthat embodiment, the tTA vector contains the tTA-encoding DNA moietyflanked by DNA sequence of the locus of the endogenous gene permittinghomologous recombination of the construct into that locus.

The use of flanking DNA sequence to permit homologous recombination intoa desired genetic locus is known in the art. At present it is preferredthat up to several kilobases or more of flanking DNA corresponding tothe chromosomal insertion site be present in the vector on both sides ofthe tTA-encoding sequence (or any other sequence of this invention to beinserted into a chromosomal location by homologous recombination) toassure precise replacement of chromosomal sequences with the exogenousDNA. See e.g. Deng et al, 1993, Mol. Cell. Biol 13(4):2134-40; Deng etal, 1992, Mol Cell Biol 12(8):3365-71; and Thomas et al, 1992, Mol CellBiol 12(7):2919-23. It should also be noted that the eucaryotic cell ofthis invention may contain multiple copies of the gene of interest, e.g.by conventional genetic amplification, each operably linked to thetTA-responsive promoter.

It should be clear from the preceding that to achieve the goals ofintroducing the DNA moiety encoding the tTA into the host cell genomeand of introducing the tTA-responsive promoter construct in operablelinkage to the desired gene, vectors based on the following principlesare required. First, to introduce the tTA-encoding construct into thegenome of the host cell such that its expression will follow theregulated pattern of expression observed in the unmodified host cell forthe gene of interest, it is necessary to introduce the tTA-encodingconstruct such that its expression is made subject to the transcriptioncontrol elements associated with the gene of interest. One way to do sois to introduce the tTA-encoding construct by homologous recombinationinto the genetic locus of the gene of interest. A vector for suchintroduction comprises the DNA sequence encoding the tTA flanked bysufficient DNA sequence from the locus of the gene of interest in thehost genome to permit the desired homologous recombination event inwhich the tTA and flanking DNA is effectively swapped for the flankingDNA copy and the DNA included there between within the host cell genome.As will be appreciated an expression construct containing a tTAresponsive promoter operably linked to the DNA sequence of theendogenous gene can be integrated at random sites without the help offlanking homologous sequences as described in references throughout thisapplication. Alternatively, to insert a DNA sequence comprising atTA-responsive promoter or tetO control element(s) upstream of a desiredgene, a construct is assembled in which the DNA comprising thetTA-responsive promoter is ligated upstream of a copy of the desiredgene between DNA sequences flanking the desired insertion site in thehost genome. In either event the tTA construct can be introduced asmentioned previously.

Using the foregoing genetic constructs and engineered eucaryotic cells,this invention further provides a method for regulating the expressionof a gene of interest. In one aspect of this method eucaryotic hostcells engineered as described above are cultured under otherwiseconventional conditions suitable for cell growth and proliferation, butin a culture medium containing a substance capable of binding to thetetracycline repressor moiety and of blocking or inhibitingtranscriptional activation. Tetracycline is the archetypical suchsubstance. However, tetracycline analogs which bind to tetR to form acomplex which is not transcriptionally activating may of course besubstituted for tetracycline. The precise concentration of tetracyclineor other such substance will depend on the substance's affinity for thetetR domain and/or the substance's specific inhibitory activity, as wellas the cell density and copy number of the tTA and the desired level ofinhibition of gene expression. Nonetheless, appropriate levels ofinhibitory substance for the desired level of inhibition are readilydeterminable empirically without undue effort.

Cell culture in accordance with the preceding method negativelyregulates, i.e. inhibits expression of the gene of interest, completelyor partially. Culturing of the cells thereafter in media with a lowerconcentration (relative to the initial concentration) of the tetRbinding substance permits gene expression to begin or to ensue at a nowhigher level. If an initial concentration of binding substance (e.g.tetracycline) is selected which is sufficient to inhibit genetranscription substantially completely (e.g. transcription is notobserved under conventional Northern blotting conditions), and in thefollowing phase of cell culture the binding substance is substantiallyremoved from the media, gene expression can be said to be regulated inan on/off manner. In some applications, intermediate levels ofexpression may be desired. To that end, concentrations of bindingsubstance may be selected based on empirical data to providepredetermined intermediate level(s) of gene transcription. It should beunderstood that removal of the binding substance from the media may beeffected by gradual, step-wise, continual or total replacement ofculture media containing the binding substance with culture medialacking the binding substance or simply containing reduced levels of thebinding substance.

Where the eucaryotic cells engineered in accordance with this inventionare incorporated into the host organism, e.g. to create a transgenicorganism, this invention provides a genetically engineered non-humananimal capable of regulatably expressing a gene of interest. Suchanimal, in the broad sense, comprises cells containing and capable ofexpressing a heterologous DNA moiety encoding a tTA as previouslydefined and a DNA moiety comprising an gene of interest under thetranscriptional control of a heterologous promoter responsive to thetranscriptional activator.

Thus, this invention further relates to non-human animals derived byhomologous recombination of one or more polynucleotide molecules of theinvention into a specific target site within their genome, the offspringof such animals, as well as to a method to prevent or promote theexpression of a targeted gene in a conditional manner.

This embodiment of the invention is able to solve a longstanding problemin the field generally described as gene targeting or gene knock out(Capecchi. M. R. (1989) Science Vol 244, p. 1288-1292, Bradley, A.(1991) Current opinion in Biotechnology Vol. 2, p. 823-829) pertainingto genes whose mutations results in death of the homozygous embryos,e.g., as described for the knock out of the RB gene (Jacks, T. et al.(1992) Nature 359:295-300). If the genetic switch subject of the currentinvention is applied as described below, expression of an endogenousgene of interest operably linked to a tet operator sequence(s) can bestimulated by a tetracycline-controllable transactivator (tTA) of theinvention and the animal develops like a nonmutated wildtype animal.Then, at a particular stage of development, expression of the endogenousgene of interest can be switched off by raising the level oftetracycline or a tetracycline analogue in the circulation and thetissues of the animal by feeding or injecting the tetracycline ortetracycline analog to the animal, thereby inhibiting the activity ofthe tTA and transcription of the gene of interst. This method isgenerally referred to herein as a "conditional knockout".

As will be clear from the following, two principally differentapproaches have been devised to apply the genetic switch of thisinvention to the genome of the non-human animal in a way, that willallow for a temporally and spatially correct expression of theendogenous gene. In one approach, the two elements of the genetic switchare in separate locations in the chromosome and require two integrationsteps, another one achieves the desired result in one step.

In the first step of one embodiment of the invention non-human animalsare derived by homologous recombination of the DNA sequences of the tTAinto a specific DNA site containing the nucleotide sequences of anendogenous gene of interest in such a way that part or all of the codingsequence of the endogenous gene is replaced with the tTA gene. This canbe accomplished (see FIG. 11) in the following steps:

(1) assembling a chimeric gene in which the sequence of the first (i.e.tTA) polynucleotide molecule of the invention is flanked by DNAsequences from the gene of interest such that upon incorporation of thechimeric gene into the host genome, the DNA sequences that normallycontrol the expression of the target gene are fused to and controlexpression of the DNA sequences for the tTA.

(2) introducing this chimeric gene into an embryonic stem cell line froma species of interest and screening resultant candidate embryonic cellclones to identify and recover those cells in which homologousrecombination has taken place at the locus of interest.

(3) introducing those recombinant cells so identified and recovered intoa blastocyst from the species of interest to yield a chimeric embryo.

(4) implanting the chimeric embryo into the uteri of pseudopregnantrecipient mothers to facilitate development and birth of a homologousrecombinant offspring.

This process results in offspring whose genome contains the DNA sequenceencoding the tTA inserted in place of the gene of interest such that thetTA DNA is expressed in a pattern similar or identical to that of thegene of interest. These processes and their results are collectively andcommonly referred to as "gene knock-out". These techniques are wellestablished and described in: Wood et al. Proc. Natl. Acad. Sci.90:4582-4585, Simon et al. Nature Genetics 1:92-97 & Soriano et al. Cell64:693-702 and references therein, the full content of which are intheir entirety incorporated herein by reference.

The second step in this embodiment of the invention relates to thepreparation of a second transgenic animal which contains in it's genomethe gene of interest under transcriptional control of the tetracycline(Tc) responsive promoter element. This can be accomplished using thefollowing method:

(1) A chimeric DNA sequence is prepared where a Tc responsive promoterelement, (comprising at least one tet operator and a minimal promoter)is cloned 5' of the DNA sequences encoding the endogenous gene ofinterest. One way to accomplish this is to replace the luciferase codingsequence and all polyadenylation elements in the plasmids pUHC13-3 orpUHC13-4 with the DNA sequence containing the complete genomic codingsequence of the endogenous gene and sufficient 3' non coding sequence toallow for proper polyadenylation. As will be appreciated the DNAsequence encoding the endogenous gene can also be cDNA (cloned as anexample in such a way that it replaces exactly the luciferase gene inpUHC13-3 or pUHC13-4) or any combination of genomic DNA and cDNAdesigned to provide the complete coding sequence, any regulatoryelements that may reside in intron sequences or is not contained in it'sentirety in the cDNA and a polyadenylation signal or other elementstypically associated with the endogenous gene. General cloning and DNAmanipulation methods are described in references cited throughout thisapplication.

(2) The chimeric DNA sequence (called also "the chimeric transgene") isinjected into a fertilized egg which is implanted into a pseudopregnantrecipient mother and allowed to develop into an adult animal. Inparticular, a few hundred DNA molecules are injected into thepro-nucleus of a fertilized one cell egg. The microinjected eggs arethen transferred into the oviducts of pseudopregnant foster mothers andallowed to develop. It has been reported by Brinster et al. (1986) Proc.Natl. Acad. Sci. USA Vol. 83:9065-9069, the full contents of which areincorporated by reference herein, that about 25% of mice which developwill inherit one or more copies of the microinjected DNA. A protocol forconstructing such transgenic animals (Brinster et al. Proc. Natl. Acad.Sci. 83:4432-4445, Crenshaw et al. Genes 3: Dev 9:959-972 and referencescited therein) is a well established technique as is the breeding ofrecombinant and hybrid animals.

Breeding of animals resulting from the first and the second step of thisembodiment of the invention produces offspring containing both thereplaced gene of interest and the chimeric transgene. In a preferredembodiment, animals heterozygous for the knockout of the endogenous generesulting from the first step of this embodiment of the invention (andinstead expressing a tTA gene) are used for breeding with animals thatare homozygous for the chimeric transgene resulting from the second stepof this embodiment of the invention. The resulting offspring areanalyzed by standard techniques, including tail-blot analysis describedin references throughout this application, and animals homozygous forboth traits are selected. Typically about 50% of the offspring shouldcarry both traits. In these animals, replacement of the coding sequencesof the gene of interest with those of the DNA sequences of the tTA issuch that the tTA is expressed in a temporal and spatial pattern similaror identical to that of the gene of interest and regulates in transexpression of the gene of interest now under transcriptional control of(i.e., operably linked to) the DNA sequences of the Tet operator andminimal promoter inserted at it's 5' end.

As will be appreciated, the particular breeding strategy depends on thenature of the gene of interest. If the "knock out" of the endogenousgene with the tTA coding sequence is not lethal and the overall plan isto create animals where the functions of the gene of interest in theadult can be studied in the "on" or "off" state, the animals from thefirst step of this embodiment of the invention can be bred tohomozygosity and then bred with the homozygous mice from the secondstep.

In this combination, the gene of interest is regulated by the additionor subtraction of tetracycline or its analogs from the food or watersupply of the animal as discussed below.

In another embodiment of the invention, embryonic stem (ES) celltechnology is used to prevent or promote expression of a gene interestin a conditional manner (FIG. 12). In the first step of this embodimentof the invention, a chimeric DNA sequence (commonly referred to as achimeric transgene) consisting of the DNA sequences of the tetoperator(s) and a suitable minimal promoter inserted 5' of the DNAsequences encoding a gene of interest is introduced by stable,non-homologous recombination into random sites in the ES cell genome.Co-introduced with this chimeric construct is a selectable marker thatenables the selection of cell clones that have integrated DNA constructsfrom cells that have not. As will be appreciated, the feeder cellssupporting the growth of the ES cells have to express the sameresistance gene used for the selection step. As an example, if theselection marker chosen is the hygromycin resistance gene, the primaryfeeder layer cells used for the ES cell culture can be derived from ananimal transgenic for the hygromycin resistance gene prepared accordingto standard procedures for the preparation of transgenic animals, ascited throughout this application. ES cell clones are selected for lowbasal expression of the chimeric transgene using customary detectionmethods, such as evaluating the mRNA levels of the transgene asdescribed in "Current Protocols in Molecular Biology" Ausubel, F. M. etal (eds.) 1989 Vol. 1 and 2 and all supplements to date, GreenePublishing Associates and Wiley-lnterscience, John Wiley & Sons, NewYork, the full contents of which are incorporated herein by reference,or as described in the other references cited throughout thisapplication. Other methods to detect expression of the transgene mayinclude activity assays or assays designed to detect protein expression.Low basal expression of the transgene is determined relative tountransfected cells. Alternatively, low basal expression of the tetoperator-linked transgene can be evaluated in different tissues ofanimals derived from the embryonic stem cells. For example, ES cells canbe transfected with the transgene in culture, and the clones expanded,selected and injected into blastocysts to create transgenic animals.After standard identification and breeding to create animals carryingthe transgene in all tissues, the baseline expression of thetet-operator linked transgene can be examined in various tissues ofinterest (e.g., by conventional techniques for analyzing mRNAexpression, such as Northern blotting, S1 nuclease mapping or reversetranscriptase-polymerase chain reaction). Additionally, basal expressionof the transgene can be examined in primary cultures of cells derivedfrom various tissues of the animal (e.g, skin cells in culture).

A second criterion for the selection of the stable clone is the abilityof the tet operator-linked transgene to respond to transient or stableexpression of tTA upon transfection of a tTA expression plasmid likepUHD 15-1 or pUHD 151-1. As will be appreciated, these plasmids arecited as examples only and others can be devised that expressedsufficient quantities of tTA in ES cells. The ability of tTA to induceexpression of a tet-operator linked transgene stably transfected into anES cell clone can be examined by supertransfecting the ES cell clonewith a tTA expression plasmid and assaying expression of the transgene.Alternatively, inducibility of a tet-operator linked transgene can beexamined in cells derived from various tissues of a transgenic animalcarrying the transgene by preparing primary cultures of cells from theanimal (e.g., skin cell cultures), transfecting the cells with a tTAexpression plasmid and assaying expression of the transgene in the cellsby standard techniques.

A clone fulfilling the criteria discussed above is selected and expandedin number. This clone is then used as a recipient of a gene knock-outprocedure consisting of the following steps:

(1) flanking the sequences of a polynucleotide molecule encoding a tTAof the invention by DNA sequences from a second gene of interest suchthat the DNA sequences that normally control the expression of thesecond target gene of interest are fused to and control expression ofthe DNA sequences of encoding the tTA;

(2) introducing this chimeric gene into an embryonic cell line from aspecies of interest and modified as described above and screeningcandidate embryonic cell clones for those in which homologousrecombination has taken place at the locus of interest;

(3) introducing those recombinant cells into blastocysts from thespecies of interest; and

(4) implanting the chimeric embryo into the uteri of pseudopregnantrecipient mothers to facilitate development and birth of a homologousrecombinant animal.

This process results in offspring containing a replacement of the aminoacid coding sequences of the second gene of interest with those of theDNA sequences of the tTA such that the tTA encoding sequence isexpressed in a temporal and spatial pattern similar to that of theendogenous second gene of interest. In this case, it is necessary toself cross the recombinant animals (or breed to homozygosity) so thatboth copies of the target sequence into which the tTA coding sequenceshave been integrated are interrupted. This procedure also leads tohomozygosity of the tet-operator linked transgene (i.e., animalshomozygous for both components of the genetic switch described hereincan be produced). These techniques are well established and describedin: Wood et al. Proc. Natl. Acad. Sci. 90:45824585, Simon et al. NatureGenetics 1:92-97; and Soriano et al. Cell 64:693-702 and referencestherein.

In yet another embodiment of the invention, embryonic stem (ES) celltechnology can again be used to prevent or promote expression of a geneinterest in a conditional manner using a single homologous recombinationstep that will result in the integrated copy shown in FIG. 13. In thismethod, a DNA construct containing a fusion of the sequences thatnormally flank the endogenous gene of interest at the 5' end (andcontain sequences commonly referred to as promoter sequences) are fusedto the DNA sequences encoding the tTA molecule. At the 3' end of the tTAcoding sequence, DNA sequences encoding resistance to a selectablemarker are typically included. For example, a neomycin resistance gene,which may be fused to either a constitutive regulatory element (e.g., apPGK promoter as depicted in FIG. 13A) or to a tet operator sequence(s)(as depicted in FIG. 13B) can be inserted at the 3' end of the tTAencoding sequence. When the selectable marker is operably linked to atet operator sequence(s), its expression is regulated by the tTA (e.g.,a resistance phenotype will be expressed in the absence but not thepresence of Tc). Finally, 3' of the selectable marker sequences in thisDNA construct are inserted the DNA sequences encoding the endogenousgene of interest, which are also fused to at least one tet operatorsequence and a minimal promoter.

Because in this configuration of the DNA molecule, conventionally calledthe targeting vector, the coding sequences of the tTA, the selectablemarker and the endogenous gene of interest are all flanked by thesequences normally flanking the endogenous gene of interest, this DNAconstruct has the potential for homologous recombination with the locusof the endogenous gene of interest upon its introduction into cells suchas, but not limited to ES cells. Homologous recombination of this typealters the natural locus such that the gene of interest falls under thecontrol of the tTA and consequently under regulation by the presence orabsence of tetracycline or derivative thereof. The expression of the tTAprotein, on the other hand, follows the normal pattern of expression ofthe gene of interest. Recombinant ES cells of this type are then used togenerate intact organisms as has been described (Wood et al. Proc. Natl.Acad. Sci. 90:4582-4585, Simon et al. Nature Genetics 1:92-97; andSoriano et al. Cell 64:693-702) which can in turn be breed tohomozygosity.

As will be appreciated, the close proximity of the promoter elements inthis particular configuration of the DNA construct used for homologousrecombination may require special consideration to insulate thedownstream tet operator/minimal promoter operably linked to theendogenous gene from long range effects of the endogenous promoteroperably linked to the tTA coding sequence to achieve the required lowbasal level expression of the endogenous gene. Some possible solutionsare strong transcriptional terminators known to those of ordinary skillin the art, DNA elements that increase the distance between the elementsor others that limit the effect of enhancer sequences (e.g.,transcriptional insulators, including matrix attachment regions), all ofwhich are to be cloned alone or in combination in between the selectablemarker expression unit (e.g., neomycin resistance gene with linkedpromoter) and the tTA-responsive transcriptional promoter sequence (seeFIG. 13). Examples of suitable transcriptional terminators,transcriptional insulators, matrix attachment regions and/or othersequences which can be included in the "single hit" targeting vector toinhibit basal transcription of the tet operator-linked endogenous geneare described in Sato, K. et al. (1986) Mol. Cell. Biol. 6:1032-1043;Michel, D. et al. (1993) Cell. Mol. Biol. Res. 39:131-140; Chung, J. H.et al. (1993) Cell 74:505-514; Neznanov, N. et al. (1993) Mol. Cell.Biol. 13:2214-2223; and Thorey, I. S. et al. (1993) Mol. Cell. Biol.13:6742-6751.

The different animals resulting from any of the above mentionedembodiments can be studied either in the absence (endogenous geneswitched "on") or presence (endogenous gene switched "off") oftetracycline or tetracycline analogues as described for other transgenicanimals below. Such animals can be used to identify, compare andcharacterize the activity of substances which interact with, upon orthrough the action of the gene product of interest.

The present invention relates to a control system that in eucaryoticcells allows regulation of expression of an individual gene over up tofive orders of magnitude. This system is based on regulatory elements ofa tetracycline resistance operon, e.g. Tn10 of E. coli (Hillen &Wissmann, "Topics in Molecular and Structural Biology," inProtein-Nucleic Acid Interaction, Saeger & Heinemann, eds., Macmillan,London, 1989, Vol. 10, pp. 143-162), in which transcription ofresistance-mediating genes is negatively regulated by the tetracyclinerepressor (tetR). In the presence of tetracycline or a tetracyclineanalogue, tetR does not bind to its operators located within thepromoter region of the operon and allows transcription. By combiningtetR with a protein capable of activating transcription in eucaryotes,e.g. the C-terminal domain of VP16 from HSV (known to be essential forthe transcription of the immediate early vital genes (Triezenberg etal., (1988) Genes Dev. 2:718-729), a hybrid transactivator is generatedthat stimulates minimal promoters fused to tetracycline operator (tetO)sequences. These promoters are virtually silent in the presence of lowconcentrations of tetracycline, which prevents thetetracycline-controlled transactivator (tTA) from binding to tetOsequences.

The specificity of the tetR for its operator sequence (Hillen &Wissmann, "Topics in Molecular and Structural Biology," inProtein-Nucleic Acid Interaction, Saeger & Heinemann, eds., Macmillan,London, 1989, Vol. 10, pp. 143-162) as well as the high affinity oftetracycline for tetR (Takahashi et al., J. Mol. Biol. 187:341-348(1986)) and the well-studied chemical and physiological properties oftetracyclines constitute a basis for an inducible expression system ineucaryotic cells far superior to the lacR/O/IPTG system. This hasalready been demonstrated in plant cells, in which direct repressoraction at promoter sites is efficiently reversed by the antibiotic (Gatz& Quail, (1988) Proc. Natl. Acad. Sci. USA 85:1394-1397, Gatz et al.,(1991) Mol. Gen. Genet. 227:229-237). However, these previous systemsused a tet repressor alone to inhibit gene expression, which may beinefficient or require high concentrations of the repressorintracellularly to function effectively. In contrast, the tTA of thepresent invention functions as a transcriptional activator to stimulateexpression of a tet operator-linked gene.

In particular, the invention relates to a polynucleotide molecule codingfor a transactivator fusion protein comprising the tet repressor (tetR)and a protein capable of directly or indirectly activating transcriptionin eucaryotes. The portion of the polynucleotide molecule coding fortetR may be obtained according to Altschmied et al., EMBO J. 7:4011-4017(1988), the contents of which are fully incorporated by referenceherein. Other tetR sequences are available from Genbank and/or aredisclosed in Waters, S. H. et al. (1983) Nucl. Acids Res. 11:6089-6105;Unger, B. et al. (1984) Gene 31:103-108, Unger, B. et al. (1984) NuclAcids Res. 12:7693-7703; Tovar, K. et al. (1988) Mol. Gen. Genet.215:76-80; Hillen, W. and Schollmeier, K. (1983) Nucl. Acids Res.11:525-539 and Postle, K. et al. (1984) Nucl. Acids Res. 12:4849-4863,the contents of each of which are fully incorporated herein byreference.

The portion of the polynucleotide molecule coding for the negativelycharged C-terminal domain of HSV-16, a protein known to be essential fortransactivation in eucaryotes, may be obtained according to Triezenberget al., Genes Dev. 2:718-729 (1988), the contents of which are fullyincorporated by reference herein. Preferably, the activating domaincomprises the C-terminal 130 amino acids of the virion protein 16.Alternatively, other polypeptides with transcriptional activationability in eucaryotic cells can be used in the tTA of the invention.Transcriptional activation domains found within various proteins havebeen grouped into categories based upon similar structural features.Types of transcriptional activation domains include acidic transcriptionactivation domains, proline-rich transcription activation domains,serine/threonine-rich transcription activation domains andglutamine-rich transcription activation domains. Examples of acidictranscriptional activation domains include the VP16 regions alreadydescribed and amino acid residues 753-881 of GAL4. Examples ofproline-rich activation domains include amino acid residues 399-499 ofCTF/NF1 and amino acid residues 31-76 of AP2. Examples ofserine/threonine-rich transcription activation domains include aminoacid residues 1-427 of ITF1 and amino acid residues 2-451 of ITF2.Examples of glutamine-rich activation domains include amino acidresidues 175-269 of Oct1 and amino acid residues 132-243 of Sp1. Theamino acid sequences of each of the above described regions, and ofother useful transcriptional activation domains, are disclosed inSeipel, K. et al. (EMBO J. (1992) 13:4961-4968).

The polynucleotide molecule coding for tetR may be linked to apolynucleotide molecule coding for the activating domain (e.g., of HSVVP16) and recombined with vector DNA in accordance with conventionalrecombinant DNA techniques, including blunt-ended or stagger-endedtermini for ligation, restriction enzyme digestion to provideappropriate termini, filling in of cohesive ends as appropriate,alkaline phosphatase treatment to avoid undesirable joining, andligation with appropriate ligases. Alternatively, nucleic acid fragmentsencoding the repressor and the activating domain can be obtained bypolymerase chain reaction amplification of appropriate nucleotidesequences using template DNA encoding either the repressor or theactivating domain (e.g., encoding VP16). The amplified DNA fragments canthen be ligated such that the protein coding sequences remain in-frameand the chimeric gene so produced can be cloned into a suitableexpression vector.

Preferably, the polynucleotide molecule coding for the transactivatorfusion protein further comprises an operably linked promoter. Thepromotor may be an inducible promoter or a constitutive promoter.Examples of such promoters include the human cytomegalovirus promoter IEas taught by Boshart et al., Cell 41:521-530 (1985), ubiquitouslyexpressing promoters such as HSV-Tk (McKnight et al., Cell 37:253-262(1984)) and β-actin promoters (e.g. the human β-actin promoter asdescribed by Ng et al., Mol. Cell. Biol. 5:2720-2732 (1985)), as well aspromoters in combination with control regions allowing integration siteindependent expression of the transgene (Grosveld et al., Cell51:975-985 (1987)), as well as tissue specific promoters such as albumin(liver specific, Pinkert et al., Genes Dev. 1:268-277 (1987)), lymphoidspecific promoters (Calame and Eaton, Adv. Immunol. 43:235-275 (1988)),in particular promoters of T-cell receptors (Winoto and Baltimore, EMBOJ. 8:729-733 (1989)) and immunoglobulins; Banerji et al., Cell33:729-740 (1983); Queen and Baltimore, ibid. 741-748), neuron specificpromoters (e.g. the neurofilament promoter; Byrne and Ruddle, Proc.Natl. Acad. Sci. USA 86:5473-5477 (1989)), pancreas specific promoters(Edlund et al., Science 230:912-916 (1985)) or mammary gland specificpromoters (milk whey promoter, U.S. Pat. No. 4,873,316 and EuropeanApplication Publication No. 264,166) as well as developmentallyregulated promoters such as the murine hox promoters (Kessel and Cruss,Science 249:374-379 (1990)) or the α-fetoprotein promoter (Campes andTilghman, Genes Dev. 3:537-546 (1989)), the contents of each of whichare fully incorporated by reference herein. Preferably, the promoter isconstitutive in the respective cell types. In one embodiment of theinvention, the polynucleotide molecule encoding the transactivator isintegrated at a predetermined location within a second target DNAmolecule (e.g., a gene of interest within a chromosome) such that thetTA-coding sequences are placed under the control of endogenousregulatory elements (e.g., a 5' regulatory region of a target gene ofinterest into which the tTA-coding sequence is integrated). Dependingupon which gene the tTA-coding sequences are integrated into, theendogenous regulatory elements may provide constitutive expression ofthe tTA in many cell types or may limit expression of the tTA to aparticular cell or tissue type.

The invention also relates to another polynucleotide molecule coding fora protein, wherein said polynucleotide is operably linked to atTA-responsive promoter. Typically, this tTA-responsive promotercomprises a minimal promoter operatively linked to at least one tetoperator (tetO) sequence. The tetO sequence may be obtained, forexample, according to Hillen & Wissmann, "Topics in Molecular andStructural Biology," in Protein-Nucleic Acid Interaction, Saeger &Heinemann, eds., Macmillan, London, 1989, Vol. 10, pp. 143-162, thecontents of which are fully incorporated by reference herein. Other tetOsequences which may be used in the practice of the invention may beobtained from Genbank and/or are disclosed in Waters, S. H. et al.(1983) Nucl. Acids Res. 11:6089-6105; Hillen, W. and Schollmeier, K.(1983) Nucl. Acids Res. 11:525-539; Stuber, D. and Bujard, H. (1981)Proc. Natl. Acad. Sci. USA 78:167-171; Unger, B. et al. (1984) NuclAcids Res. 12:7693-7703; and Tovar, K. et al. (1988) Mol. Gen. Genet.215:76-80, which are fully incorporated by reference herein in theirentirety. One, two, three, four, five, six, seven, eight, nine or ten ormore copies of the tet operator sequence may be employed, with a greaternumber of such sequences allowing an enhanced range of regulation. Asshown in the Examples, multiple copies of the tet operator sequenceprovides a synergistic effect on the ability to control expression ofthe heterologous protein.

The polynucleotide sequence specifying the cytomegalovirus promoter maybe obtained according to Boshart et al., Cell 41:521-530 (1985), thecontents of which are fully incorporated by reference herein.Preferably, positions +75 to -53 to +75 to -31 of the promoter-enhancerare employed as a minimal promoter. The promoter may be followed by apolylinker and then by the gene coding for the protein of interest.While the luciferase gene or other reporter gene, e.g. the gene codingfor chloramphenicol acetyltransferase or β-galactosidase, may be used todemonstrate the operability of the regulatory system, the invention isnot intended to be so limited. Examples of such genes include, but arenot limited to the estrogen receptor, the GABA receptor, theprogesterone receptor and the X-protein of HBV.

The present invention also relates to eucaryotic cells transfected withthe polynucleotide molecules of the present invention. In particular,the invention relates to eucaryotic cells transfected with

(a) a first polynucleotide molecule coding for a transactivator fusionprotein comprising a prokaryotic tet repressor and a protein capable ofactivation transcription in eucaryotes; and

(b) a second polynucleotide molecule coding for a protein, wherein saidsecond polynucleotide molecule is operably linked to a minimal promoterand at least one tet operator sequence.

The two polynucleotide molecules may reside on the same or separatevectors. In a preferred embodiment, the first polynucleotide isintegrated into the chromosome of a eucaryotic cell or transgenic animaland the second polynucleotide is introduced as part of a vector.Integration may be achieved where there is crossover at regions ofhomology shared between the incoming polynucleotide molecule and theparticular genome.

The expression of the heterologous protein from such transfectedeucaryotic cells may be tightly regulated. Unexpectedly, it has beendetermined that the expression system of the present invention may beused to regulate expression by about 5 orders of magnitude. In addition,it has been discovered that the expression system of the presentinvention allows one to rapidly turn "on" and "off" the expression ofthe heterologous gene in a reversible way. Moreover, it has beendiscovered that the expression system of the invention allows one toachieve a desired level of expression according to how much tetracyclineor tetracycline analogue is employed (see FIG. 3). Thus, the expressionsystem of the present invention is a great advance in the art.

The invention also relates to transgenic animals comprising at least afirst polynucleotide molecule of the present invention encoding a tTA.Such transgenic animals may be obtained, for example, by injecting thepolynucleotide into a fertilized egg which is allowed to develop into anadult animal. In particular, a few hundred DNA molecules are injectedinto the pro-nucleus of a fertilized one cell egg. The microinjectedeggs are then transferred into the oviducts of pseudopregnant fostermothers and allowed to develop. It has been reported by Brinster et al.,Proc. Natl. Acad. Sci. USA 82:4438-4442 (1985), the contents of whichare fully incorporated by reference herein, that about 25% of mice whichdevelop will inherit one or more copies of the microinjected DNA. It isalso possible to prepare a polynucleotide molecule comprising a milkprotein promotor and microinject the DNA into the fertilized egg togive, upon development, a transgenic mammal which is capable ofproducing the heterologous protein in its milk, when in the absence oftetracycline or a tetracycline analog. See International ApplicationPublication No. WO 88/00239 and European Application Publication No.0264,166, the contents of which are fully incorporated by referenceherein.

The invention also relates to non-human animals and their offspringderived by homologous recombination of the DNA sequences of the firstpolynucleotide molecules of the invention into a specific DNA sitecontaining the nucleotide sequences of a gene referred to as the targetgene. This would be accomplished in the following steps: 1) flanking thesequences of the first polynucleotide molecule of the invention encodinga tTA by DNA sequences from the target site such that the DNA sequencesthat normally control the expression of the target gene are fused to andcontrol the expression of the DNA sequences of the first polynucleotidemolecules of the invention, 2) introducing this chimeric gene into anembryonic cell line from the species of interest and screening candidateembryonic cell clones for those in which homologous recombination hastaken place at the target gene locus, 3) introducing those recombinantcells into a blastocysts from the species of interest, 4) implanting thechimeric embryo into the uteri of pseudopregnant recipient mothers tofacilitate development and birth. This process will result in offspringcontaining a replacement of the amino acid coding sequences of thetarget gene with those of the DNA sequences of the first polynucleotidemolecule of the invention such that this corresponding amino acidsequence will be expressed in a pattern similar to that of the targetgene. These processes and their results are collectively and commonlyreferred to as "gene knock-out". These techniques are well establishedand described in: Wood et al. Proc. Natl. Acad Sci. 90:4582-4585, Simonet al. Nature Genetics 1:92-97 & Soriano et al. Cell 64:693-702 andreferences therein.

The invention also relates to a method to prevent or promote theexpression of the target gene in a conditional manner. This may beaccomplished by breeding an animal containing the target gene knock-out(as outlined in the preceding paragraph) with a transgenic animalderived by the following method. The transgenic animal would beconstructed by inserting, by micro-injection, a chimeric DNA sequence(commonly referred to as a chimeric transgene) consisting of the DNAsequences of the second polynucleotide molecule of the inventioninserted 5' of the DNA sequences encoding the amino acid sequence of thetarget gene into the genome of a fertilized egg which is allowed todevelop into an adult animal. The protocol for the construction of suchtransgenic animals is a well established technique (Brinster et al.Proc. Natl. Acad. Sci. 83:4432-4445, Crenshaw et al. Genes & Dev3:959-972 and references therein) as is the breeding of animals. Fromthe breeding will result offspring containing both the gene knock-outand the chimeric transgene. That is, replacement of the amino acidcoding sequences of the target gene with those of the DNA sequences ofthe first polynucleotide molecule of the invention such that thiscorresponding amino acid sequence will be expressed in a pattern similarto that of the target gene and, the DNA sequences of the secondpolynucleotide molecule of the invention inserted 5' of the DNAsequences encoding the amino acid sequence of the target gene. In thiscombination the target gene can be regulated by the addition orsubtraction of tetracycline or its analogs from the food or water supplyof the animal.

Thus, the invention also relates to a method to down regulate theexpression of a protein coded for by a polynucleotide, comprisingcultivating the transfected eucaryotic cells of the present invention ina medium comprising tetracycline or a tetracycline analogue. Asdescribed in the Examples, it is possible to closely control the extentof expression by carefully controlling the concentration of tetracyclineor tetracycline analogue in the culture media. As shown in FIG. 3, panelA, as little as 0.0001 μg/ml of tetracycline will begin to result in adecrease of polypeptide (luciferase) expression. At about 0.1 μg/ml, theexpression is essentially shut off. The concentration of tetracycline ortetracycline analogue which can be used to regulate the expression levelmay range from about 0.001 to about 1 μg/ml.

The invention also relates to a method to up regulate the expression ofa protein coded for by a polynucleotide, comprising cultivating theeucaryotic cells of the present invention in a medium lackingtetracycline or a tetracycline analogue.

The invention also relates to a method to use regulated gene expressionin the production of recombinant proteins as generally reviewed byYarranton, G. T 1992, the whole article incorporated as referenceherein. Expression of recombinant proteins that are cytotoxic orotherwise infer with physiological processes in cells has been hamperedby the lack of suitable methods to tightly regulate gene expression. Incontrast, a production cell line according to the current invention isgrown in the presence of tetracycline or tetracycline analogues until anoptimal density (assessed empirically to allow for subsequent inductionof gene expression) and expression is induced by dilution of theregulating compound. The culture is continuously grown until an optimalexpression level has been reached. The recombinant protein is thenharvested according to standard procedures.

As a preferred embodiment, eucaryotic cells are used for expression ofrecombinant proteins as generally reviewed in "Gene Transfer andExpression" (M. Kriegler 1990) incorporated herein as reference. WhileCHO^(dhfr-) cells (Urlaub, G. and Chasin, L. 1980), 293 cells (Graham,F. L. et al. 1977) or myeloma cells like SP2 or NS0 (Galfre, C. andMilstein, C. 1981) are commonly used it should be clear to the skilledin the art, that any eucaryotic cell line can be used that is suitablefor the protein to be expressed, the selection system chosen and thefermentation system employed.

In another preferred embodiment, the cells used for regulated expressionare yeast cells including, but not limited to Saccharomyces cerevisiae,Pichia pastoris, Kluyveromyces lactis and Hansenula polymorpha asgenerally reviewed by Fleer, R. 1992, the whole article incorporated asreferenced herein.

In another preferred embodiment, the cells used for regulated expressionare insect cells with the gene and promoter region carried on thebaculovirus genome as generally reviewed in "Baculovirus expressionvectors" (O'Reilly et al. 1992), the whole document incorporated asreferenced herein.

As can be appreciated, the tissue specificity of some promoters dictatethat the tet operator sequence/promoter sequence fusion has to bedesigned with the particular application and cell line in mind followingthe teachings in this application using the promoters customarily usedfor the cell line in question; examples for those promoters are given inthe relevant references mentioned above.

It should be clear from the foregoing that it is critical in the currentinvention that the production cell line is selected for a very low basalexpression of the gene under control of the Tet operator/CMV promotersequence. There are numerous methods currently available employingenzymatically assisted or unassisted homologous recombination to targetrepeatedly a chromosomal location found empirically to be suited for theintegration of the gene encoding the recombinant protein. In addition tothe homologous recombination approaches already described herein,enzyme-assisted site-specific integration systems are known in the artand can be applied to the components of the regulatory system of theinvention to integrate a DNA molecule at a predetermined location in asecond target DNA molecule. Examples of such enzyme-assisted integrationsystems include the Cre recombinase-lox target system (e.g., asdescribed in Baubonis, W. and Sauer, B. (1993) Nucl. Acids Res.21:2025-2029; and Fukushige, S. and Sauer, B. (1992) Proc. Natl. Acad.Sci. USA 89:7905-7909) and the FLP recombinase-FRT target system (e.g.,as described in Dang, D. T. and Perrimon, N. (1992) Dev. Genet.13:367-375; and Fiering, S. et al. (1993) Proc. Natl. Acad. Sci. USA90:8469-8473).

Media which may be used in the practice of the invention include anymedia which are compatible with the transfected eucaryotic cells of thepresent invention. Such media are commercially available (e.g. fromGibco/BRL).

Alternatively, it is possible to down regulate the expression of aprotein in a transgenic animal of the present invention by administeringto the animal tetracycline or tetracycline analogue. The tetracycline ortetracycline may be administered by any means that achieves its intendedpurpose, e.g. by parenteral, subcutaneous, intravenous, intramuscular,intraperitoneal, transdermal, or buccal routes. Alternatively, orconcurrently, administration may be by the oral route (see e.g., Example2). The dosage administered will be dependent upon the age, health, andweight of the animal, kind of concurrent treatment, if any, andfrequency of treatment. To up regulate the expression of the protein,the administration of tetracycline or tetracycline analogue may then beinterrupted.

The invention also relates to a kit comprising a carrier means having inclose confinement therein at least two container means such as tubes,vials, bottles and the like, each of which containing a polynucleotidemolecule which can be used in the practice of the invention. Inparticular, the invention relates to a kit comprising a carrier meanshaving in close confinement therein at least two container means,wherein a first container means contains a first polynucleotide moleculecoding for a transactivator fusion protein comprising a prokaryotic tetrepressor and a protein capable of activation transcription ineucaryotes in a form suitable for homologous recombination; and a secondcontainer means contains a second polynucleotide molecule comprising aminimal promoter operably linked to at least one tet operator sequence,wherein the second polynucleotide molecule is capable of being ligatedto a heterologous gene sequence coding for a polypeptide and activatingthe expression of the heterologous protein.

The invention also relates to kits comprising a carrier means having inclose confinement therein at least two container means, wherein a firstcontainer means contains a eucaryotic cell transfected with a firstpolynucleotide molecule coding for a transactivator fusion proteincomprising a prokaryotic tet repressor and a protein capable ofactivation transcription in eucaryotes in a form suitable for homologousrecombination; and a second container means contains a secondpolynucleotide molecule comprising a minimal promoter operably linked toat least one tet operator sequence, wherein the second polynucleotidemolecule is capable of being ligated to a heterologous gene sequencecoding for a polypeptide and activating expression of the polypeptide.

The invention is widely applicable to a variety of situations where itis desirable to be able to turn gene expression "on" and "off", orregulate the level of gene expression, in a rapid, efficient andcontrolled manner without causing pleiotropic effects or cytotoxicity.The invention may be particularly useful for gene therapy purposes inhumans, in treatments for either genetic or acquired diseases. Thegeneral approach of gene therapy involves the introduction of one ormore nucleic acid molecules into cells such that one or more geneproducts encoded by the introduced genetic material are produced in thecells to restore or enhance a functional activity. For reviews on genetherapy approaches see Anderson, W. F. (1992) Science 256:808-813;Miller, A. D. (1992) Nature 357:455-460; Friedmann, T. (1989) Science244:1275-1281; and Cournoyer, D., et al. (1990) Curr. Opin. Biotech.1:196-208. However, current gene therapy vectors typically utilizeconstitutive regulatory elements which are responsive to endogenoustranscriptions factors. These vector systems do not allow for theability to modulate the level of gene expression in a subject. Incontrast, the regulatory system of the invention provides this ability.

To use the system of the invention for gene therapy purposes, at leastone DNA molecule is introduced into cells of a subject in need of genetherapy (e.g., a human subject suffering from a genetic or acquireddisease) to modify the cells. The cells are modified to contains 1)nucleic acid encoding a tTA of the invention in a form suitable forexpression of the tTA in the host cells and 2) a gene of interest (e.g.,for therapeutic purposes) operatively linked to a tTA-responsivepromoter (e.g., a tet operator sequence(s) and minimal promoter).Preferably, one or both of these DNA molecules is integrated into apredetermined location within a chromosome of the human cells byhomologous recombination. A single DNA molecule encoding both componentsof the regulatory system of the invention can be used, or alternatively,separate DNA molecules encoding each component can be used. The cells ofthe subject can be modified ex vivo and then introduced into the subjector the cells can be directly modified in vivo by conventional techniquesfor introducing nucleic acid into cells. Expression of the gene ofinterest in the cells of the subject is stimulated in the absence of Tcor a Tc analogue, whereas expression is then inhibited by administeringTc or a Tc analogue to the patient. The level of gene expression can bevaried depending upon which particular Tc analogue is used as theinducing agent. Additionally, expression of the gene of interest can beadjusted according to the medical needs of the individual, which mayvary throughout the lifetime of the individual. Thus, the regulatorysystem of the invention offers the advantage over constitutiveregulatory systems of allowing for modulation of the level of geneexpression depending upon the requirements of the therapeutic situation.

Genes of particular interest to be expressed in cells of a subject fortreatment of genetic or acquired diseases include those encodingadenosine deaminase, Factor VIII, Factor IX, dystrophin, β-globin, LDLreceptor, CFTR, insulin, erythropoietin, anti-angiogenesis factors,growth hormone, glucocerebrosidase, β-glucouronidase, α1-antitrypsin,phenylalanine hydroxylase, tyrosine hydroxylase, ornithinetranscarbamylase, arginosuccinate synthetase, UDP-glucuronysyltransferase, apoA1, MDR1 and MRP multidrug resistance genes, TNF,soluble TNF receptor, interleukins (e.g., IL-2), interferons (e.g., α-or γ-IFN) and other cytokines and growth factors.

Gene therapy applications of particular interest in cancer treatmentinclude overexpression of a cytokine gene (e.g., TNF-α) in tumorinfiltrating lymphocytes or ectopic expression of cytokines in tumorcells to induce an anti-tumor immune response at the tumor site),expression of an enzyme in tumor cells which can convert a non-toxicagent into a toxic agent, expression of tumor specific antigens toinduce an anti-tumor immune response, expression of tumor suppressorgenes (e.g., p53 or Rb) in tumor cells, expression of a multidrugresistance gene (e.g., MDR1 and/or MRP) in bone marrow cells to protectthem from the toxicity of chemotherapy.

Gene therapy applications of particular interest in treatment of viraldiseases include expression of trans-dominant negative viraltransactivation proteins, such as trans-dominant negative tat and revmutants for HIV or trans-dominant ICp4 mutants for HSV (see e.g.,Balboni, P. G. et al. (1993) J. Med. Virol. 41:289-295; Liem, S. E. etal. (1993) Hum. Gene Ther. 4:625-634; Malim, M. H. et al. (1992) J. Exp.Med. 176:1197-1201; Daly, T. J. et al. (1993) Biochemistry 32:8945-8954;and Smith, C. A. et al. (1992) Virology 191:581-588), expression oftrans-dominant negative envelope proteins, such as env mutants for HIV(see e.g., Steffy, K. R. et al. (1993) J. Virol. 67:1854-1859),intracellular expression of antibodies, or fragments thereof, directedto viral products ("internal immunization", see e.g., Marasco, W. A. etal. (1993) Proc. Natl. Acad. Sci. USA 90:7889-7893) and expression ofsoluble viral receptors, such as soluble CD4.

The regulatory system of the invention can also be used to express asuicide gene (such as a ricin or HSV tk gene) in cells in a conditionalmanner to allow for destruction of the cells (e.g., in vivo) following aparticular therapy. For example, a suicide gene can be introduced intotumor cells to be used for anti-cancer immunization or into the viralgenome of a live attenuated viral to be used as a vaccine. The tumorcells or viral vaccine carrying the suicide gene are administered to asubject in the presence of Tc (or analogue thereof). Followingimmunization, the drug is withdrawn (e.g., administration is stopped),thereby inducing expression of the suicide gene to destroy the tumorcells or cells carrying the live virus.

Cells types which can be modified for gene therapy purposes includehematopoietic stem cells, myoblasts, hepatocytes, lymphocytes, airwayepithelium and skin epithelium. For further descriptions of cell types,genes and methods for gene therapy see e.g., Wilson, J. M et al. (1988)Proc. Natl. Acad. Sci. USA 85:3014-3018; Armentano, D. et al. (1990)Proc. Natl. Acad. Sci. USA 87:6141-6145; Wolff, J. A. et al. (1990)Science 247:1465-1468; Chowdhury, J. R. et al. (1991) Science254:1802-1805; Ferry, N. et al. (1991) Proc. Natl. Acad. Sci. USA88:8377-8381; Wilson, J. M. et al. (1992) J. Biol. Chem. 267:963-967;Quantin, B. et al. (1992) Proc. Natl. Acad. Sci. USA 89:2581-2584; Dai,Y. et al. (1992) Proc. Natl. Acad. Sci. USA 89:10892-10895; vanBeusechem, V. W. et al. (1992) Proc. Natl. Acad Sci. USA 89:7640-7644;Rosenfeld, M. A. et al. (1992) Cell 68:143-155; Kay, M. A. et al. (1992)Human Gene Therapy 3:641-647; Cristiano, R. J. et al. (1993) Proc. Natl.Acad Sci. USA 90:2122-2126; Hwu, P. et al. (1993) J. Immunol.150:4104-4115; and Herz, J. and Gerard, R. D. (1993) Proc. Natl. AcadSci. USA 90:2812-2816.

The regulatory system of the invention can also be used to produce andisolate a gene product (e.g., protein) of interest. Large scaleproduction of a protein of interest can be accomplished using culturedcells in vitro which have been modified to contain 1) nucleic acidencoding a tTA of the invention in a form suitable for expression of thetTA in the host cells and 2) a gene of interest (e.g., encoding aprotein of interest) operatively linked to a tTA-responsive promoter(e.g., a tet operator sequence(s) and minimal promoter). For example,mammalian, yeast or fungal cells can be modified to contain thesenucleic acid components as described herein. Alternatively, an insectcell/baculovirus expression system can be used. To produce and isolate agene product of interest, a host cell (e.g., mammalian, yeast or fungalcell) carrying the two components of the regulatory system of theinvention (e.g., nucleic acid encoding a tTA and a gene of interest,encoding the gene product of interest, linked to a tTA-responsivepromoter) are first grown in a culture medium in the presence oftetracycline or a tetracycline analogue. Under these conditions,expression of the gene of interest is repressed. Next, the concentrationof tetracycline or the tetracycline analogue in the culture medium isreduced to stimulate transcription of the gene of interest. The cellsare then further cultured in the absence of Tc (or analogue thereof)until a desired amount of the gene product encoded by the gene ofinterest is produced by the cells. The gene product can then be isolatedfrom harvested cells or from the culture medium by standard techniques.

The invention also provides for large scale production of a protein ofinterest in animals, such as in transgenic farm animals. Advances intransgenic technology have made it possible to produce transgeniclivestock, such as cattle, goats, pigs and sheep (reviewed in Wall, R.J. et al. (1992) J. Cell. Biochem. 49:113-120; and Clark, A. J. et al.(1987) Trends in Biotechnology 5:20-24). Accordingly, transgeniclivestock carrying in their genome the components of the regulatorysystem of the invention can be constructed. Thus, by appropriate mating,double transgenic animals carrying a transgene encoding a tTA of theinvention and a transgene comprising a tTA-responsive promoter linked toa gene of interest (the gene of interest may be either an exogenous oran endogenous gene) can be obtained. In the absence of Tc (or analogue),expression of the gene of interest is stimulated in the transgenicanimals. By administering Tc (or analogue) to the animal, expression ofthe gene of interest can be inhibited. Protein production can betargeted to a particular tissue by linking the nucleic acid encoding thetTA to an appropriate tissue-specific regulatory element(s) which limitsexpression of the transactivator to certain cells. For example, amammary gland-specific regulatory element, such as the milk wheypromoter (U.S. Pat. No. 4,873,316 and European Application PublicationNo. 264,166), can be linked to the tTA-encoding transgene to limitexpression of the transactivator to mammary tissue. Thus, in the absenceof Tc (or analogue), the protein of interest will be produced in themammary tissue of the transgenic animal, whereas protein expression canbe downmodulated by administering Tc or a Tc analogue. The protein canbe designed to be secreted into the milk of the transgenic animal, andif desired, the protein can then be isolated from the milk.

Having now generally described this invention, the same will beunderstood by reference to the following examples which are providedherein for purposes of illustration only and are not intended to belimiting unless otherwise specified. The contents of all publications,references, patents and published patent applications cited throughoutthe application are hereby incorporated by reference.

EXAMPLE 1 Regulation of Gene Expression in Cells by tTA

Materials and Methods

Construction of the transactivators tTA and tTA_(S). The tetR sequencewas originally recovered from pWH510 (Altschmied et al., EMBO J.7:4011-4017 (1988), the disclosure of which is fully incorporated byreference herein) by PCR and inserted into pUHD10-1 (Deustchle et al.,Proc. Natl. Acad. Sci. USA 86:5400-5404 (1989)), resulting in pUHD14-1(see, the Dissertation of Manfred Gossen, "Prokaryotic RepressorOperator Systems in the Control of Eucaryotic Gene Expression,Heidelberg University, 1993, the contents of which are fullyincorporated by reference herein). A unique AflII cleavage site,overlapping the tetR stop codon in this plasmid construct, allows forthe in-frame insertion of coding sequences. To generate tTA, a397-base-pair (bp) MluI/FokI fragment of pMSVP16 (Triezenberg et al.,Genes Dev. 2:718-729 (1988), the disclosure of which is fullyincorporated by reference herein), coding for the C-terminal 130 aminoacids of VP16 of HSV, was blunted by filling in the protruding ends withT4 DNA polymerase. This DNA was inserted in pUHD14-1, previously cleavedwith AfllI, and blunted by mung bean nuclease. The resulting plasmid,pUHD15-1, encodes the tTA sequence (FIG. 1, panel a) under the controlof the P_(hCMV) (human cytomegalovirus promoter IE; see below). In ahomologous approach, a DNA fragment coding for the 97-amino acidC-terminal portion of VP16 was fused to tetR by PCR-mediated cloning.The resulting plasmid, pUHD151-1, encodes the smaller version of thetrans-activator, tTA_(S) (FIG. 1, panel a).

Construction of P_(hCMV) * and the Luciferase Reporter Plasmid.

Plasmid pUHC13-1 is a derivative of pUHD10-1 (Deuschle et al., Proc.Natl. Acad. Sci. USA 86:5400-5404 (1989)). It contains thepromoter-enhancer sequence of PhCMV, spanning position +75 to position-675 (Boshart et al., Cell 41:521-530 (1985)). This promoter is followedby a polylinker and the luciferase gene of Photinus pyralis fused to theSV40 small-t intron and poly(A) signal. The latter elements and theluciferase gene were transferred from pSV2L,AΔ5' (DeWit et al., Mol.Cell. Biol. 7:725-737 (1987)). By this transfer, the N-terminus ofluciferase has been modified as described (Deuschle et al., Proc. Natl.Acad. Sci. USA 86:5400-5404 (1989)). The enhancer region of P_(hCMV) wasremoved by PCR-mediated cloning, whereby a Xho I site was introducedadjacent to position -53. The resulting minimal promoter, P_(hCMV) *(FIG. 1, panel b) is part of the reporter plasmid pUHC13-2.

Construction of P_(hCMV) *-1 and P_(hCMV) *-2.

To combine P_(hCMV) * with tet operators, the 19-bp inverted repeatsequence of operator 02 of Tn10 (Triezenberg et al., Genes Dev.2:718-729 (1988)) was synthesized as part of a 42-bp DNA fragment [SEQID NO: 10]: (upper strand: 5' TCGAGTTTACCACTCCCTATCAGTGATAGAGAAAAGTGAAAG3'). Upon annealing, the two complementary strands exposed thecompatible protruding ends of a Xho I and a Sal I cleavage site at the 5and 3' ends, respectively. Ligation of this fragment into the Xho I siteof the polylinker of pT81-luc (Nordeen, S. K., BioTechniques 6:454-457(1988)) created, upon cloning, single as well as multiple inserts ofoperator sequences upstream of a thymidine kinase (tk) minimal promoterfrom HSV contained in pT81-luc. The tk promoters containing one, two,and seven operator sequences were examined for their ability to beactivated in transient expression experiments using the HeLa cell lineHtTa-1 (see below). All constructs were active in tTA producing cells ina tetracycline-dependent manner. The heptameric version of the tetOsequences caused by far the highest activation of all Ptk-tetOconstructs. It therefore was removed as a Xhol/Sal fragment andtransferred into pUHC13-2. Due to the asymmetric location of the tetOwithin the polylinker of pT81-luc, the resulting plasmids pUHC13-3 andpUHC13-4 contain the heptameric tetOs in two orientations differing inthe distance between the operators and position +1 of P_(hCMV) by 19 bp.The two tetO-containing promoters were designated P_(hCMV) *-1 andP_(hCMV) *-2 (FIG. 1, panel b).

Band-Shift Assay.

Cytoplasmic and nuclear cell extracts from ˜2×10⁶ cells were prepared asdescribed by Andrews and Faller, Nucl. Acids Res. 19:2499 (1991), exceptthat the cytoplasmic protein fraction was centrifuged once more (1 hr,100,000× g). Nuclear proteins were extracted by a buffer containing 20mM Hepes-KOH (pH 7.9), 25% glycerol, 420 mM NaCL, 1.5 mM MgCl₂, 0.2 mMEDTA, 0.5 mM dithiothreitol, and 0.5 mM phenylmethylsulfonyl fluoride.Aliquots (5 μl) of nuclear extracts were mixed with 15 μl of bindingbuffer (10 mM Tris HCl, pH 7.5/10 mM MgCl₂) containing 20 μg of calfthymus DNA, 5 μg of bovine serum albumin, and 2 fmol of ³² P-labeledtetO DNA. The tetO DNA was isolated from pUHC13-3 as a 42-bp Taq Ifragment whose protruding ends were filled in by Klenow enzyme in thepresence of [α-³² P)dCTP. After 20 min. at room temperature, aliquots ofthe binding reaction mixture were loaded onto a 5% polyacrylamide/0.07%bisacrylamide gel. Electrophoresis was carried out in 90 mM Tris base/90mM boric acid/3 mM EDTA at 5 V/cm.

Luciferase Assays.

Cell grown to ˜80% confluency in 35-mm dishes in Eagle's minimumessential medium were washed with 2 ml of phosphate-buffered salinebefore they were lysed in 25 mM Tris phosphate, pH 7.8/2 mMdithiothreitol/2 mM diaminocyclohexanetetraacetic acid/10% glycerol/1%Triton X-100 for 10 min at room temperature. The lysate was scraped offthe culture dishes and centrifuged for 10 sec in an Eppendorfcentrifuge. Next, aliquots (10 μl) of the supernatant were mixed with250 μl of 25 mM glycylglycine/15 mM MgSO₄ /5 mM ATP and assayed forluciferase activity in a Lumat LB9501 (Berthold, Wildbad, F. R. G.)using the integral mode (10 sec). D-Luciferin (L6882, Sigma) was used at0.5 mM. The background signal measured in extracts of HeLa cells thatdid not contain a luciferase gene was indistinguishable from theinstrumental background [80-120 relative light units (rlu)/10 sec).Protein content of the lysates was determined according to Bradford(Bradford, M. M., Anal. Biochem. 72:248-254 (1976)).

RESULTS

Construction and Characterization of the tTA.

To convert the prokaryotic tet repressor into a eucaryotictransactivator, it was fused to the negatively charged C-terminal domainof HSV-VP16, known to be essential for transactivation. (Triezenberg etal., Genes Dev. 2:718-729 (1988)). Sequences coding for either a 97- ora 127-amino acid C-terminal portion of VP16 were fused to the tetR gene,resulting in the coding sequences of tTAS and tTA, respectively (FIG. 1,panel a). In plasmids coding for tTA (pUHD15-tTAs (pUHD151-1), thetransactivator sequences are flanked upstream by P_(hCMV) and downstreamby the SV40 poly(A) site. The two fusion proteins did not differ intheir functional in vivo properties.

HeLa cells transiently transfected with pUHD15-1 produced a fusionprotein of the expected molecular mass (37 kDa), as demonstrated inimmunoblots of the electrophoretically separated cytoplasmic and nuclearextracts (FIG. 2, panel a). When nuclear extracts were mixed with thetetO DNA, the electrophoretic mobility of the DNA was diminished. Thespecificity of the interaction between tTA and operator DNA wasconfirmed by the finding that no mobility change for tetO DNA wasdetectable in the presence of the specific inducer tetracycline (FIG. 2,panel b).

Construction of a tTA-Dependent Promoter.

To generate promoters activatable by tTA, tetOs were inserted upstreamof minimal promoter sequences. For P_(hCMV), the upstream enhancerregion was removed by PCR and a Xho I cleavage site was introducedadjacent to position -53. This minimal promoter, designated P_(hCMV) *,spans the original P_(hCMV) sequence from +75 to -53 (+1 being the firstnucleotide transcribed) and, in addition, contains a Stu I site around-31 (FIG. 1, panel b). tetO sequences were fused to this core promoterby insertions at the Xho I site (FIG. 1 ).

The tetO sequence 02 of Tn10 is a 19-bp inverted repeat to which tetRbinds as a 46-kDa dimer (Hillen & Wissmann, "Topics in Molecular andStructural Biology," in Protein-Nucleic Acid Interaction, Saeger&˜Heinemann, eds., Macmillan, London, 1989, Vol. 10, pp. 143-162). Itwas chemically synthesized and ligated into the Xho I cleavage site ofthe polylinker located upstream of the minimal tk promoter in plasmidpT81-luc (Nordeen, S. K., BioTechniques 6:454-457 (1988)). Multipleinsertions of tetOs created a set of promoters that contained between 1and 7 tetO sequences upstream from position -81 of the tk promoter. AXho l/Sal I fragment containing 7 tetOs, fused head to tail, wasrecovered from one of the constructs and transferred into the Xho I siteupstream of P_(hCMV) *. Due to the asymmetry of the Xho l/Sal Ifragment, two P_(hCMV) *-tetO constructs were obtained that differ inthe distance between the operators and position +1 of P_(hCMV), which is95 bp for P_(hCMV) *-1 and 76 bp for P_(hCMV) *-2. The plasmidscontaining these promoters are designated pUHC13-3 and pUHC13-4,respectively (FIG. 1, panel b). When HeLa cells were transientlytransfected with these plasmids, high levels of luciferase activity weremonitored whenever the cells were cotransfected with pUHD15-1, whichprovided the coding sequence of tTA. Little activity was observed withcultures grown in the presence of tetracycline (1.0 μg/ml) or withplasmids containing P_(hCMV) * only. Since P_(hCMV) *-1 and P_(hCMV) *-2were activated by tTA to a significantly higher degree than any of thePtk constructs, the latter ones were not investigated further.

Quantitation of P_(hCMV) *-1 and P_(hCMV) *-2 Activation by tTA.

To quantify the stimulation of P_(hCMV) *-tetO constructs by tTA, HeLacell lines were established that contained the P_(hCMV) *-1- or theP_(hCMV) *-2-luciferase, as well as the P_(hCMV) -tTA expression unitsstably integrated. Conditions for culturing and selecting cells havebeen described (Deuschle et al., Proc. Natl. Acad. Sci. USA 86:5400-5405(1989)). In a first step, cells were cotransfected with pUHD15-1 andpSV2neo (Southern &: Berg, J. Mol. Appl. Genet. 1:327-341 (1982)).Clones resistant to G418 were assayed for transactivation of P_(hCMV)*-1 by transient transfection with pUHC13-3. In all HeLa cell clones inwhich the tetracycline-responsive promoters were active, tTA was notdetectable by Western blots or by immunofluorescence. Its presence wasjust barely visible in electrophoretic mobility shift experiments ofhighly labeled tetO DNA. This indicates very low intracellularconcentrations of tTA and may reflect a selection against squelchingeffects caused by higher concentrations of VP16-activating domains (Gill& Ptashne, Nature (London) 334:721-724 (1988).

One of the positive clones, HtTA-1, was then cotransfected with aplasmid carrying the hygromycin-resistance gene (pHMR272; Bernard etal., Exp. Cell Res. 158:237-243 (1985)) and either pUHC13-3 or pUHC13-4,resulting in the X and T series of clones, respectively. Clonesresistant to hygromycin and G418 were assayed for luciferase activity.As shown in Table 1 below, in the absence of tetracycline, this activitydiffered in individual clones by almost four orders of magnitude.However, in all cases, the luciferase activity was sensitive totetracycline in the culture. This demonstrates that the expression ofluciferase is dependent on the function of tTA, which obviously iscapable of activating promoter constructs P_(hCMV) *-1 and P_(hCMV) *-2.

                  TABLE 1                                                         ______________________________________                                        Tetracycline-dependent Luciferase Activity of                                 Different HeLa Cell Clones                                                    Luciferase activity, rlu/μ of protein                                      Clone  With Tc     Without Tc  Activation Factor                              ______________________________________                                        T7     1074 ± 75                                                                              79,197 ± 2,119                                                                         7.3 × 10.sup.1                           T11    2.5 ± 0.4                                                                              34,695 ± 1,127                                                                         1.3 × 10.sup.4                           T12    3.5 ± 0.9                                                                              35,298 ± 5,009                                                                           1 × 10.sup.4                           T14    <2          33 ± 4   ≧1.5 × 10.sup.1                   T15    286 ± 47 49,070 ± 2,784                                                                         1.7 × 10.sup.2                           T16    <2           541 ± 133                                                                             ≧2.7 × 10.sup.2                   X1     <2          257,081 ± 40,137                                                                       ≧2.7 × 10.sup.5                   X2     <2          104,840 ± 20,833                                                                       ≧5 × 10.sup.4                     X7     75 ± 7   125,745 ± 18,204                                                                       1.6 × 10.sup.3                           ______________________________________                                         The HeLa cell clone HtTA1, which constitutively expresses tTA, was            cotransfected with pUHC133 or pUHC134 and pHMR272. Hygromycinresistant        clones were examined for luciferase activity. Nine clones identified were     subcloned and luciferase activity was quantified in the presence (            μ/ml) and absence of tetracycline (Tc). Values are arithmetic means of     three independent luciferase determinations (from three independently         grown cultures). Luciferase activities of <2 rlu/μg of protein are too     close to the instrumental background to be quantified.                   

When the luciferase activity within various clones was monitored in thepresence and absence of tetracycline hydrochloride (Sigma), tworemarkable results emerged. (i) In all clones tested, tTA greatlystimulated promoter activity, even up to five orders of magnitude inclone X1. (ii) In clones T14, T16, X1 and X2 (Table 1), tetracyclinereduced luciferase activity to values that cannot be quantified even athigh protein concentration of extracts due to instrumental limitations(i.e., rlu/μg of protein>2). This demonstrates that P_(hCMV) *-1 andP_(hCMV) *-2 are virtually silent when integrated in the proper genomicenvironment and that their activity depends exclusively on the action oftTA.

The tTA inactivation studies were carried out with 1 μg of tetracyclineper ml in the culture medium. A partial inactivation of tTA is, however,readily achieved with tetracycline concentrations below 0.1 μg/ml, asshown in FIG. 3, panel a. In the two clones analyzed (T12 and X1), astepwise reduction of the tetracycline concentration in the mediumgradually increased the luciferase activity. These results againdemonstrate that, in the case of clone X1, tTA can regulatetranscriptional activity, as monitored by luciferase activity, by overfive orders of magnitude. Moreover, at tetracycline concentrationssufficient for full inactivation of tTA (0.1 μg/ml), no change in growthbehavior or morphology of HeLa cells occurs. Only at tetracyclineconcentrations well above 10 μg/ml were such changes observed uponprolonged incubation.

Kinetics of Tetracycline Action.

The time course of tetracycline action was analyzed in cultures grown inthe absence or presence of tetracycline. At time 0, the antibiotic wasadded to the tetracycline-free cultures (final concentration, 1 μg/ml),whereas the tetracycline-containing cultures were rinsed and incubatedin fresh antibiotic-free medium (FIG. 3 panel b). At various times,cells were harvested and analyzed for luciferase activity. As shown inFIG. 3 panel b, the depletion of tetracycline leads to a rapid inductionof luciferase activity reaching >20% of the fully induced level within12 hr. A similarly rapid reduction of luciferase activity was observedwhen tetracycline was added to the fully active tetracycline-freesystem: within 8 hr activity dropped to about 10% and reached <2% of itsoriginal value after 12 hr.

The fusion of the Tn10-derived E. coli tetR with the activation domainof VP16 from HSV has generated a transactivator exhibiting all of theproperties required for the specific and stringent regulation of anindividual gene in a eucaryotic cell. The transactivator tTA produced inHeLa cells binds specifically to tetO sequences in vitro. Thisassociation is prevented by tetracycline. When bound to tetOs placedupstream of minimal promoters, tTA efficiently activates transcriptionfrom such promoters in vivo in a tetracycline-dependent manner. Thetransactivator is produced in HeLa cells in amounts sufficiently highfor strong activation of transcription though low enough to avoid anydetectable squelching effects (Gill & Ptashne Nature (London)334:721-724 (1988)).

The usefulness of heterologous regulatory systems as the one describedhere depends decisively on quantitative parameters such as the extent ofinactivation and the efficiency of activation of gene expression as wellas the kinetics of transition from one state to the other. For the tetsystem, these parameters were measured in HeLa cell lines thatconstitutively express tTA and that also contain the luciferase genestably integrated and under the control of tTA-dependent promoters. Theclones characterized thus far express the luciferase gene to variousextents. This is not surprising since differences in the integrationsites and in the number of integrated transcription units would beexpected. However, in all cases, the expression of luciferase issensitive to tetracycline. In some clones, tetracycline has the mostdramatic effect of reducing the luciferase activity from high levelsover several orders to magnitude to background. This demonstrates thatin HeLa cells, the two promoters P_(hCMV) *-1 and P_(hCMV) *-2, have nomeasurable intrinsic activity. Their function strictly depends on tTA.The residual luciferase activity observed in some clones in the presenceof tetracycline must therefore be due to position effects.

The tTA-dependent promoters can be kept in a partially activated stateby low concentrations of tetracycline. As shown in FIG. 3 panel a,varying the tetracycline concentration between 0 and 0.1 μg/ml allowsadjustment of promoter activity within a range of several orders ofmagnitude. This may allow assessment also of quantitative parameters ofgene function in vivo.

The activation and inactivation of tTA by the antibiotic appears to benot only an efficient but also a rapid process. When cells fromtetracycline containing medium are shifted to tetracycline-free medium,significant luciferase activity is induced within 4 hr and >20% of thesteady-state level is reached within 12 hr after the shift.Interestingly, even the cultures that were only exposed totetracycline-free medium during the washing procedure beforereincubation in tetracycline-containing medium show a small butreproducible increase in luciferase activity that is still detectableafter 4 hr (FIG. 3b).

When tetracycline is added to a culture of X1 cells, luciferase activityis reduced ˜10-fold within 8 hr and >50 fold within 12 hr. This decreaseis remarkably fast if one takes into account the half-life of luciferaseof around 3 hr reported for eucaryotic cells (measured by cycloheximideinhibition: Ilguyen et al., J. Biol. Chem. 264:10487-10492 (1989);Thompson et al., Gene 103:171-177 (1991)) and indicates a rapid uptakeof tetracycline by HeLa cells followed by a fast and efficient shutdownof transcription. Although the half-life of luciferase and its mRNAremains to be determined in this system, these conclusions are supportedby observations in plant cells, where tetracycline inactivates tetRwithin <30 min (Gatz et al., Mol. Gen. Genet 227:229-237 (1991)).

Taken together, these data show that tetracycline, unlike IPTG in aeucaryotic lacR/O-based system, is able to act fast in cultures ofeucaryotic cells. The possibility of rapidly switching the activity of atTA-dependent promoter not only is of interest in studying gene functionitself but also should allow analysis of mRNA decay rates of individualgenes under physiological conditions.

In clone X1, tetracycline reduces luciferase activity reproducibly byfive orders of magnitude. This suggests that binding of tetracycline totTA may lower the association constant between the transactivator andits operator to a much greater extent than that measured for tetR(Takahasi et al., J. Mol. Biol. 187:341-348 (1986)) and as described forIPTG in the lacR/O system, where the binding constant k_(RO) is reducedonly 1000-fold by the inducer (Barkley and Bourgeois in The Operon,Miller and Reznikoff (eds.), Cold Spring Harbor Laboratory, Cold SpringHarbor, N.Y., 1980; pp. 177-220.)

On the other hand, the results obtained in transient experiments withminimal tk promoters fused to single, dimeric, and heptameric tetOsequences strongly suggest a synergistic effect of multiple tTA bindingsites. The efficient inactivation of tTA by tetracycline is thereforemost likely due to a large difference in the binding constants of tTAand tTA/tetracycline for the tetO and the nonlinear effect oftetracycline interfering with a cooperative process.

In conclusion, the results indicate that promoter-activating systems asdescribed here are most promising for regulating individual genes inhigher eucaryotic cells for several reasons. (i) For activators, inparticular when acting through a cooperative mechanism, intracellularconcentrations can be kept low, ensuring an efficient inactivation bythe effector--in this case, tetracycline. By contrast, repressors ingeneral complete directly with a transcription factors and/or RNApolymerases for binding within a promoter region. In the absence ofcooperativity, however, the window at which the repressor concentrationis sufficiently high for tight expression but still low enough forefficient induction may be narrow and not easily adjustable in differentsystems. (ii) In an activating system, as described here, the synthesisof tTA can be driven by a tissue-specific promoter, whereas the tTAdependent promoters are expected to function tissue independently, sincethey may require only general transcription factors in addition to tTA.By contrast, in a repressor-based system in which operators have to beplaced within the context of a promoter sequence, an influence onpromoter specificity cannot be excluded. (iii) The tet system offersspecific advantages when compared to the intensely studied lac system.For example, tetR binds tetracycline much tighter (ka˜10⁹ M⁻¹ ;Takahashi et al., J. Mol. Biol. 187:341-348 (1986)) than lacR complexesIPTG (ka 10⁶ M⁻¹ ; Barkley & Bourgeois in The Operon, Miller &Rezinkoff, eds., Cold Spring Harbor Lab., Cold Spring Harbor, N.Y.,1980, pp. 177-220). Thus, very low, nontoxic concentrations oftetracycline function effectively. Moreover, a large number oftetracycline analogues are known, of which some appear to have farsuperior properties as effectors than tetracycline itself. In thiscontext, it is interesting to note that detailed information on thepharmacological properties of tetracycline, in particularpharmacokinetic parameters, is available, which will facilitateapplication of this system in transgenic animals.

EXAMPLE 2 Regulation of Gene Expression in Transgenic Animals by tTA

To examine the ability of tTA to regulate gene expression in vivo,transgenic strains of mice were constructed which contained heterologouschromosomal insertions of either a tTA expression unit or atTA-responsive promoter operably linked to a reporter gene. Singletransgenic strains containing either the tTA expression unit or thetTA-responsive reporter unit were then cross bred and double transgenicprogeny were identified. The double transgenic animals were thencharacterized as to the ability of tTA, in a tetracycline dependentmanner, to regulate expression of the reporter gene. This exampledemonstrates that tTA effectively stimulates the expression of a geneoperably linked to a tTA responsive promoter in multiple tissues of theanimals in vivo in the absence of tetracycline (or analogue), whereasexpression of the tTA-responsive gene is effectively inhibited inmultiple tissues of the animals when tetracycline or an analogue thereofis administered to the animals. These results demonstrate that thetetracycline-controlled transcriptional regulatory system describedherein functions effectively in animals, in addition to cell lines invitro.

Generation of mice transgenic for a P_(hCMV) -tTA expression unit

Mice expressing tTA protein were obtained by pronuclear injection intofertilized oocytes of a 2.7 kb XhoI-PfmI fragment excised from plasmidpUHG15-1. This DNA fragment contained the tTA gene (shown in SEQ IDNO: 1) under the transcriptional control of the human CMV IE promoter(position +75 to -675) together with a rabbit β-globin polyadenylationsite including an intron. The human CMV IE promoter is a constitutivepromoter that allows expression of the tetR-VP16 fusion protein in manycell lines where chromosomal integration of the DNA sequence encodingtTA has occurred and is known to be functional in a variety of tissuesin transgenic mice. DNA was injected into fertilized oocytes at aconcentration of approximately 5 ng per μl by standard techniques.Transgenic mice were generated from the injected fertilized oocytesaccording to standard procedures. Transgenic founder mice were analyzedusing polymerase chain reaction (PCR) and Southern hybridization todetect the presence of the tTA transgene in chromosomal DNA of the mice.

Generation of mice transgenic for the P_(hCMV) *-1 luciferase reporterunit

Mice carrying a P_(hCMV) *-1 luc reporter gene expression unit weregenerated by pronuclear injection into fertilized oocytes of a 3.1 kbXhoI-EaeI fragment excised from plasmid pUHC13-3. This DNA-fragmentcontains the luciferase gene under transcriptional control of thetetracycline-responsive P_(hCMV) *-1 promoter (SEQ ID NO: 5), togetherwith a SV40 t early polyadenylation site including an intron. DNA wasinjected into oocytes at a concentration of approximately 5 ng per μland transgenic mice were generated according to standard procedures.Transgenic founder mice were analyzed using Southern hybridization todetect the presence of the P_(hCMV) *-1 luc transgene in chromosomal DNAof the mice.

Generation of mice transgenic for the P_(hCMV) *-1 luc and P_(hCMV) tTA

Having constructed single transgenic mice expressing tTA or carryingP_(hCMV) *-1 luc, double transgenic mice carrying both the tTAexpression vector and the luciferase reporter-units were obtainedthrough cross breeding of heterozygous mice transgenic for one of thetwo transgenes. Double transgenic animals were identified by standardscreenings (e.g., PCR and/or Southern hybridization) to detect thepresence of both the tTA transgene and the P_(hCMV) *-1 luc transgene inchromosomal DNA of the mice.

Induction and analysis of luciferase activity in tissue samples frommice

For oral administration, tetracycline or its derivative doxycycline weregiven in the drinking water at a concentration of 200 μg per ml with 5%sucrose to hide the bitter taste of the antibiotics. For lactating mice,the concentration was 2 mg per ml with 10% sucrose to ensure asufficient uptake via the milk by the young.

To analyze luciferase activity, mice were killed by cervical dislocationand tissue samples were homogenized in 2 ml tubes containing 500 μllysis-buffer (25 mM Tris phosphate, pH 7.8/2 mM DTT/2 mM EDTA/10%glycerol/1% Triton X100) using a Ultra-Turrax. The homogenate was frozenin liquid nitrogen and centrifuged after thawing for 5 min at 15,000 g.2-20 μl of the supernatant were mixed with 250 μl luciferase assaybuffer (25 mM glycylglycine, pH 7.5/15 mM MgSO4/5 mM ATP) and luciferaseactivity was measured for 10 sec after the injection of 100 μl of a 125μM luciferin solution using Berthold Lumat LB 9501. The proteinconcentration of the homogenate was determined using Bradford assay andluciferase activity was calculated as relative light units (rlu) per μgof total protein.

Results

Mice from 4 lines carrying the P_(hCMV) -tTA transgene (CT1 through CT4)were mated with mice from line L7, transgenic for P_(hCMV) *-1 luc. Thisline shows a very low but significant background of luciferase activityin different organs that is probably due to position effects at theintegration side. The luciferase activity in different tissues of thedouble transgenic mice, either in the presence or absence of thetetracycline analogue doxycycline, is illustrated graphically in FIG.14. High luciferase activity was detectable in five tissues of thedouble transgenic mice examined: heart, muscle, pancreas, thymus andtongue. The tissue pattern of activated luciferase levels (i.e., in theabsence of doxycycline) in the double transgenic mice was similar toexpression patterns of the hCMV IE promoter reported in the literature.This is consistent with expression of the luc reporter gene beingregulated by tTA (which is expressed in the mice under the control ofthe hCMV IE promoter). After administration of doxycycline to the micefor 7 days, luciferase activity was reduced close to background levelsobserved in single transgenic mice carrying only the P_(hCMV) *-1 lucreporter unit (i.e., the L7 line). Depending on the individual animalsused for comparison of induced and non-induced luciferase level,regulation factors up to 10,000 fold can be estimated e.g. in thepancreas. These results indicate that the tetracycline-controlledtranscription regulatory system described herein can be used toefficiently regulate expression of genes in transgenic animals.

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 10                                                 (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1008 base pairs                                                   (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Herpes Simplex Virus                                            (B) STRAIN: K12, KOS                                                          (vii) IMMEDIATE SOURCE:                                                       (B) CLONE: tTA transactivator                                                 (ix) FEATURE:                                                                 (A) NAME/KEY: exon                                                            (B) LOCATION: 1..1008                                                         (ix) FEATURE:                                                                 (A) NAME/KEY: mRNA                                                            (B) LOCATION: 1..1008                                                         (ix) FEATURE:                                                                 (A) NAME/KEY: misc. binding                                                   (B) LOCATION: 1..207                                                          (ix) FEATURE:                                                                 (A) NAME/KEY: misc. binding                                                   (B) LOCATION: 208..335                                                        (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 1..1005                                                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       ATGTCTAGATTAGATAAAAGTAAAGTGATTAACAGCGCATTAGAGCTG48                            MetSerArgLeuAspLysSerLysValIleAsnSerAlaLeuGluLeu                              151015                                                                        CTTAATGAGGTCGGAATCGAAGGTTTAACAACCCGTAAACTCGCCCAG96                            LeuAsnGluValGlyIleGluGlyLeuThrThrArgLysLeuAlaGln                              202530                                                                        AAGCTAGGTGTAGAGCAGCCTACATTGTATTGGCATGTAAAAAATAAG144                           LysLeuGlyValGluGlnProThrLeuTyrTrpHisValLysAsnLys                              354045                                                                        CGGGCTTTGCTCGACGCCTTAGCCATTGAGATGTTAGATAGGCACCAT192                           ArgAlaLeuLeuAspAlaLeuAlaIleGluMetLeuAspArgHisHis                              505560                                                                        ACTCACTTTTGCCCTTTAGAAGGGGAAAGCTGGCAAGATTTTTTACGT240                           ThrHisPheCysProLeuGluGlyGluSerTrpGlnAspPheLeuArg                              65707580                                                                      AATAAGGCTAAAAGTTTTAGATGTGCTTTACTAAGTCATCGCGATGGA288                           AsnLysAlaLysSerPheArgCysAlaLeuLeuSerHisArgAspGly                              859095                                                                        GCAAAAGTACATTTAGGTACACGGCCTACAGAAAAACAGTATGAAACT336                           AlaLysValHisLeuGlyThrArgProThrGluLysGlnTyrGluThr                              100105110                                                                     CTCGAAAATCAATTAGCCTTTTTATGCCAACAAGGTTTTTCACTAGAG384                           LeuGluAsnGlnLeuAlaPheLeuCysGlnGlnGlyPheSerLeuGlu                              115120125                                                                     AATGCATTATATGCACTCAGCGCTGTGGGGCATTTTACTTTAGGTTGC432                           AsnAlaLeuTyrAlaLeuSerAlaValGlyHisPheThrLeuGlyCys                              130135140                                                                     GTATTGGAAGATCAAGAGCATCAAGTCGCTAAAGAAGAAAGGGAAACA480                           ValLeuGluAspGlnGluHisGlnValAlaLysGluGluArgGluThr                              145150155160                                                                  CCTACTACTGATAGTATGCCGCCATTATTACGACAAGCTATCGAATTA528                           ProThrThrAspSerMetProProLeuLeuArgGlnAlaIleGluLeu                              165170175                                                                     TTTGATCACCAAGGTGCAGAGCCAGCCTTCTTATTCGGCCTTGAATTG576                           PheAspHisGlnGlyAlaGluProAlaPheLeuPheGlyLeuGluLeu                              180185190                                                                     ATCATATGCGGATTAGAAAAACAACTTAAATGTGAAAGTGGGTCCGCG624                           IleIleCysGlyLeuGluLysGlnLeuLysCysGluSerGlySerAla                              195200205                                                                     TACAGCCGCGCGCGTACGAAAAACAATTACGGGTCTACCATCGAGGGC672                           TyrSerArgAlaArgThrLysAsnAsnTyrGlySerThrIleGluGly                              210215220                                                                     CTGCTCGATCTCCCGGACGACGACGCCCCCGAAGAGGCGGGGCTGGCG720                           LeuLeuAspLeuProAspAspAspAlaProGluGluAlaGlyLeuAla                              225230235240                                                                  GCTCCGCGCCTGTCCTTTCTCCCCGCGGGACACACGCGCAGACTGTCG768                           AlaProArgLeuSerPheLeuProAlaGlyHisThrArgArgLeuSer                              245250255                                                                     ACGGCCCCCCCGACCGATGTCAGCCTGGGGGACGAGCTCCACTTAGAC816                           ThrAlaProProThrAspValSerLeuGlyAspGluLeuHisLeuAsp                              260265270                                                                     GGCGAGGACGTGGCGATGGCGCATGCCGACGCGCTAGACGATTTCGAT864                           GlyGluAspValAlaMetAlaHisAlaAspAlaLeuAspAspPheAsp                              275280285                                                                     CTGGACATGTTGGGGGACGGGGATTCCCCGGGTCCGGGATTTACCCCC912                           LeuAspMetLeuGlyAspGlyAspSerProGlyProGlyPheThrPro                              290295300                                                                     CACGACTCCGCCCCCTACGGCGCTCTGGATATGGCCGACTTCGAGTTT960                           HisAspSerAlaProTyrGlyAlaLeuAspMetAlaAspPheGluPhe                              305310315320                                                                  GAGCAGATGTTTACCGATCCCCTTGGAATTGACGAGTACGGTGGGTAG1008                          GluGlnMetPheThrAspProLeuGlyIleAspGluTyrGlyGly                                 325330335                                                                     (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 335 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       MetSerArgLeuAspLysSerLysValIleAsnSerAlaLeuGluLeu                              151015                                                                        LeuAsnGluValGlyIleGluGlyLeuThrThrArgLysLeuAlaGln                              202530                                                                        LysLeuGlyValGluGlnProThrLeuTyrTrpHisValLysAsnLys                              354045                                                                        ArgAlaLeuLeuAspAlaLeuAlaIleGluMetLeuAspArgHisHis                              505560                                                                        ThrHisPheCysProLeuGluGlyGluSerTrpGlnAspPheLeuArg                              65707580                                                                      AsnLysAlaLysSerPheArgCysAlaLeuLeuSerHisArgAspGly                              859095                                                                        AlaLysValHisLeuGlyThrArgProThrGluLysGlnTyrGluThr                              100105110                                                                     LeuGluAsnGlnLeuAlaPheLeuCysGlnGlnGlyPheSerLeuGlu                              115120125                                                                     AsnAlaLeuTyrAlaLeuSerAlaValGlyHisPheThrLeuGlyCys                              130135140                                                                     ValLeuGluAspGlnGluHisGlnValAlaLysGluGluArgGluThr                              145150155160                                                                  ProThrThrAspSerMetProProLeuLeuArgGlnAlaIleGluLeu                              165170175                                                                     PheAspHisGlnGlyAlaGluProAlaPheLeuPheGlyLeuGluLeu                              180185190                                                                     IleIleCysGlyLeuGluLysGlnLeuLysCysGluSerGlySerAla                              195200205                                                                     TyrSerArgAlaArgThrLysAsnAsnTyrGlySerThrIleGluGly                              210215220                                                                     LeuLeuAspLeuProAspAspAspAlaProGluGluAlaGlyLeuAla                              225230235240                                                                  AlaProArgLeuSerPheLeuProAlaGlyHisThrArgArgLeuSer                              245250255                                                                     ThrAlaProProThrAspValSerLeuGlyAspGluLeuHisLeuAsp                              260265270                                                                     GlyGluAspValAlaMetAlaHisAlaAspAlaLeuAspAspPheAsp                              275280285                                                                     LeuAspMetLeuGlyAspGlyAspSerProGlyProGlyPheThrPro                              290295300                                                                     HisAspSerAlaProTyrGlyAlaLeuAspMetAlaAspPheGluPhe                              305310315320                                                                  GluGlnMetPheThrAspProLeuGlyIleAspGluTyrGlyGly                                 325330335                                                                     (2) INFORMATION FOR SEQ ID NO:3:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 894 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Herpes Simplex Virus                                            (B) STRAIN: K12, KOS                                                          (C) INDIVIDUAL ISOLATE: tTAS transactivator                                   (ix) FEATURE:                                                                 (A) NAME/KEY: exon                                                            (B) LOCATION: 1..894                                                          (ix) FEATURE:                                                                 (A) NAME/KEY: mRNA                                                            (B) LOCATION: 1..894                                                          (ix) FEATURE:                                                                 (A) NAME/KEY: misc. binding                                                   (B) LOCATION: 1..207                                                          (ix) FEATURE:                                                                 (A) NAME/KEY: misc. binding                                                   (B) LOCATION: 208..297                                                        (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 1..891                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                       ATGTCTAGATTAGATAAAAGTAAAGTGATTAACAGCGCATTAGAGCTG48                            MetSerArgLeuAspLysSerLysValIleAsnSerAlaLeuGluLeu                              151015                                                                        CTTAATGAGGTCGGAATCGAAGGTTTAACAACCCGTAAACTCGCCCAG96                            LeuAsnGluValGlyIleGluGlyLeuThrThrArgLysLeuAlaGln                              202530                                                                        AAGCTAGGTGTAGAGCAGCCTACATTGTATTGGCATGTAAAAAATAAG144                           LysLeuGlyValGluGlnProThrLeuTyrTrpHisValLysAsnLys                              354045                                                                        CGGGCTTTGCTCGACGCCTTAGCCATTGAGATGTTAGATAGGCACCAT192                           ArgAlaLeuLeuAspAlaLeuAlaIleGluMetLeuAspArgHisHis                              505560                                                                        ACTCACTTTTGCCCTTTAGAAGGGGAAAGCTGGCAAGATTTTTTACGT240                           ThrHisPheCysProLeuGluGlyGluSerTrpGlnAspPheLeuArg                              65707580                                                                      AATAACGCTAAAAGTTTTAGATGTGCTTTACTAAGTCATCGCGATGGA288                           AsnAsnAlaLysSerPheArgCysAlaLeuLeuSerHisArgAspGly                              859095                                                                        GCAAAAGTACATTTAGGTACACGGCCTACAGAAAAACAGTATGAAACT336                           AlaLysValHisLeuGlyThrArgProThrGluLysGlnTyrGluThr                              100105110                                                                     CTCGAAAATCAATTAGCCTTTTTATGCCAACAAGGTTTTTCACTAGAG384                           LeuGluAsnGlnLeuAlaPheLeuCysGlnGlnGlyPheSerLeuGlu                              115120125                                                                     AATGCATTATATGCACTCAGCGCTGTGGGGCATTTTACTTTAGGTTGC432                           AsnAlaLeuTyrAlaLeuSerAlaValGlyHisPheThrLeuGlyCys                              130135140                                                                     GTATTGGAAGATCAAGAGCATCAAGTCGCTAAAGAAGAAAGGGAAACA480                           ValLeuGluAspGlnGluHisGlnValAlaLysGluGluArgGluThr                              145150155160                                                                  CCTACTACTGATAGTATGCCGCCATTATTACGACAAGCTATCGAATTA528                           ProThrThrAspSerMetProProLeuLeuArgGlnAlaIleGluLeu                              165170175                                                                     TTTGATCACCAAGGTGCAGAGCCAGCCTTCTTATTCGGCCTTGAATTG576                           PheAspHisGlnGlyAlaGluProAlaPheLeuPheGlyLeuGluLeu                              180185190                                                                     ATCATATGCGGATTAGAAAAACAACTTAAATGTGAAAGTGGGTCTGAT624                           IleIleCysGlyLeuGluLysGlnLeuLysCysGluSerGlySerAsp                              195200205                                                                     CCATCGATACACACGCGCAGACTGTCGACGGCCCCCCCGACCGATGTC672                           ProSerIleHisThrArgArgLeuSerThrAlaProProThrAspVal                              210215220                                                                     AGCCTGGGGGACGAGCTCCACTTAGACGGCGAGGACGTGGCGATGGCG720                           SerLeuGlyAspGluLeuHisLeuAspGlyGluAspValAlaMetAla                              225230235240                                                                  CATGCCGACGCGCTAGACGATTTCGATCTGGACATGTTGGGGGACGGG768                           HisAlaAspAlaLeuAspAspPheAspLeuAspMetLeuGlyAspGly                              245250255                                                                     GATTCCCCGGGTCCGGGATTTACCCCCCACGACTCCGCCCCCTACGGC816                           AspSerProGlyProGlyPheThrProHisAspSerAlaProTyrGly                              260265270                                                                     GCTCTGGATATGGCCGACTTCGAGTTTGAGCAGATGTTTACCGATGCC864                           AlaLeuAspMetAlaAspPheGluPheGluGlnMetPheThrAspAla                              275280285                                                                     CTTGGAATTGACGAGTACGGTGGGTTCTAG894                                             LeuGlyIleAspGluTyrGlyGlyPhe                                                   290295                                                                        (2) INFORMATION FOR SEQ ID NO:4:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 297 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                       MetSerArgLeuAspLysSerLysValIleAsnSerAlaLeuGluLeu                              151015                                                                        LeuAsnGluValGlyIleGluGlyLeuThrThrArgLysLeuAlaGln                              202530                                                                        LysLeuGlyValGluGlnProThrLeuTyrTrpHisValLysAsnLys                              354045                                                                        ArgAlaLeuLeuAspAlaLeuAlaIleGluMetLeuAspArgHisHis                              505560                                                                        ThrHisPheCysProLeuGluGlyGluSerTrpGlnAspPheLeuArg                              65707580                                                                      AsnAsnAlaLysSerPheArgCysAlaLeuLeuSerHisArgAspGly                              859095                                                                        AlaLysValHisLeuGlyThrArgProThrGluLysGlnTyrGluThr                              100105110                                                                     LeuGluAsnGlnLeuAlaPheLeuCysGlnGlnGlyPheSerLeuGlu                              115120125                                                                     AsnAlaLeuTyrAlaLeuSerAlaValGlyHisPheThrLeuGlyCys                              130135140                                                                     ValLeuGluAspGlnGluHisGlnValAlaLysGluGluArgGluThr                              145150155160                                                                  ProThrThrAspSerMetProProLeuLeuArgGlnAlaIleGluLeu                              165170175                                                                     PheAspHisGlnGlyAlaGluProAlaPheLeuPheGlyLeuGluLeu                              180185190                                                                     IleIleCysGlyLeuGluLysGlnLeuLysCysGluSerGlySerAsp                              195200205                                                                     ProSerIleHisThrArgArgLeuSerThrAlaProProThrAspVal                              210215220                                                                     SerLeuGlyAspGluLeuHisLeuAspGlyGluAspValAlaMetAla                              225230235240                                                                  HisAlaAspAlaLeuAspAspPheAspLeuAspMetLeuGlyAspGly                              245250255                                                                     AspSerProGlyProGlyPheThrProHisAspSerAlaProTyrGly                              260265270                                                                     AlaLeuAspMetAlaAspPheGluPheGluGlnMetPheThrAspAla                              275280285                                                                     LeuGlyIleAspGluTyrGlyGlyPhe                                                   290295                                                                        (2) INFORMATION FOR SEQ ID NO:5:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 450 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Human cytomegalovirus                                           (B) STRAIN: K12, Towne                                                        (ix) FEATURE:                                                                 (A) NAME/KEY: mRNA                                                            (B) LOCATION: 382..450                                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                       GAATTCCTCGAGTTTACCACTCCCTATCAGTGATAGAGAAAAGTGAAAGTCGAGTTTACC60                ACTCCCTATCAGTGATAGAGAAAAGTGAAAGTCGAGTTTACCACTCCCTATCAGTGATAG120               AGAAAAGTGAAAGTCGAGTTTACCACTCCCTATCAGTGATAGAGAAAAGTGAAAGTCGAG180               TTTACCACTCCCTATCAGTGATAGAGAAAAGTGAAAGTCGAGTTTACCACTCCCTATCAG240               TGATAGAGAAAAGTGAAAGTCGAGTTTACCACTCCCTATCAGTGATAGAGAAAAGTGAAA300               GTCGAGCTCGGTACCCGGGTCGAGTAGGCGTGTACGGTGGGAGGCCTATATAAGCAGAGC360               TCGTTTAGTGAACCGTCAGATCGCCTGGAGACGCCATCCACGCTGTTTTGACCTCCATAG420               AAGACACCGGGACCGATCCAGCCTCCGCGG450                                             (2) INFORMATION FOR SEQ ID NO:6:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 450 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Human cytomegalovirus                                           (B) STRAIN: Towne                                                             (ix) FEATURE:                                                                 (A) NAME/KEY: mRNA                                                            (B) LOCATION: 382..450                                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                       GAATTCCTCGACCCGGGTACCGAGCTCGACTTTCACTTTTCTCTATCACTGATAGGGAGT60                GGTAAACTCGACTTTCACTTTTCTCTATCACTGATAGGGAGTGGTAAACTCGACTTTCAC120               TTTTCTCTATCACTGATAGGGAGTGGTAAACTCGACTTTCACTTTTCTCTATCACTGATA180               GGGAGTGGTAAACTCGACTTTCACTTTTCTCTATCACTGATAGGGAGTGGTAAACTCGAC240               TTTCACTTTTCTCTATCACTGATAGGGAGTGGTAAACTCGACTTTCACTTTTCTCTATCA300               CTGATAGGGAGTGGTAAACTCGAGTAGGCGTGTACGGTGGGAGGCCTATATAAGCAGAGC360               TCGTTTAGTGAACCGTCAGATCGCCTGGAGACGCCATCCACGCTGTTTTGACCTCCATAG420               AAGACACCGGGACCGATCCAGCCTCCGCGG450                                             (2) INFORMATION FOR SEQ ID NO:7:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 398 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Herpes Simplex Virus                                            (B) STRAIN: KOS                                                               (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                       GAGCTCGACTTTCACTTTTCTCTATCACTGATAGGGAGTGGTAAACTCGACTTTCACTTT60                TCTCTATCACTGATAGGGAGTGGTAAACTCGACTTTCACTTTTCTCTATCACTGATAGGG120               AGTGGTAAACTCGACTTTCACTTTTCTCTATCACTGATAGGGAGTGGTAAACTCGACTTT180               CACTTTTCTCTATCACTGATAGGGAGTGGTAAACTCGACTTTCACTTTTCTCTATCACTG240               ATAGGGAGTGGTAAACTCGACTTTCACTTTTCTCTATCACTGATAGGGAGTGGTAAACTC300               GAGATCCGGCGAATTCGAACACGCAGATGCAGTCGGGGCGGCGCGGTCCGAGGTCCACTT360               CGCATATTAAGGTGACGCGTGTGGCCTCGAACACCGAG398                                     (2) INFORMATION FOR SEQ ID NO:8:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 6244 base pairs                                                   (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: circular                                                        (ii) MOLECULE TYPE: DNA (genomic)                                             (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Human cytomegalovirus                                           (B) STRAIN: Towne (hCMV)                                                      (vii) IMMEDIATE SOURCE:                                                       (B) CLONE: pUHD BGR3                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                                       CTCGAGTTTACCACTCCCTATCAGTGATAGAGAAAAGTGAAAGTCGAGTTTACCACTCCC60                TATCAGTGATAGAGAAAAGTGAAAGTCGAGTTTACCACTCCCTATCAGTGATAGAGAAAA120               GTGAAAGTCGAGTTTACCACTCCCTATCAGTGATAGAGAAAAGTGAAAGTCGAGTTTACC180               ACTCCCTATCAGTGATAGAGAAAAGTGAAAGTCGAGTTTACCACTCCCTATCAGTGATAG240               AGAAAAGTGAAAGTCGAGTTTACCACTCCCTATCAGTGATAGAGAAAAGTGAAAGTCGAG300               CTCGGTACCCGGGTCGAGTAGGCGTGTACGGTGGGAGGCCTATATAAGCAGAGCTCGTTT360               AGTGAACCGTCAGATCGCCTGGAGACGCCATCCACGCTGTTTTGACCTCCATAGAAGACA420               CCGGGACCGATCCAGCCTCCGCGGCCCCGAATTCGAGCTCGGTACCGGGCCCCCCCTCGA480               GGTCGACGGTATCGATAAGCTTGATATCGAATTCCAGGAGGTGGAGATCCGCGGGTCCAG540               CCAAACCCCACACCCATTTTCTCCTCCCTCTGCCCCTATATCCCGGCACCCCCTCCTCCT600               AGCCCTTTCCCTCCTCCCGAGAGACGGGGGAGGAGAAAAGGGGAGTTCAGGTCGACATGA660               CTGAGCTGAAGGCAAAGGAACCTCGGGCTCCCCACGTGGCGGGCGGCGCGCCCTCCCCCA720               CCGAGGTCGGATCCCAGCTCCTGGGTCGCCCGGACCCTGGCCCCTTCCAGGGGAGCCAGA780               CCTCAGAGGCCTCGTCTGTAGTCTCCGCCATCCCCATCTCCCTGGACGGGTTGCTCTTCC840               CCCGGCCCTGTCAGGGGCAGAACCCCCCAGACGGGAAGACGCAGGACCCACCGTCGTTGT900               CAGACGTGGAGGGCGCATTTCCTGGAGTCGAAGCCCCGGAGGGGGCAGGAGACAGCAGCT960               CGAGACCTCCAGAAAAGGACAGCGGCCTGCTGGACAGTGTCCTCGACACGCTCCTGGCGC1020              CCTCGGGTCCCGGGCAGAGCCACGCCAGCCCTGCCACCTGCGAGGCCATCAGCCCGTGGT1080              GCCTGTTTGGCCCCGACCTTCCCGAAGACCCCCGGGCTGCCCCCGCTACCAAAGGGGTGT1140              TGGCCCCGCTCATGAGCCGACCCGAGGACAAGGCAGGCGACAGCTCTGGGACGGCAGCGG1200              CCCACAAGGTGCTGCCCAGGGGACTGTCACCATCCAGGCAGCTGCTGCTCCCCTCCTCTG1260              GGAGCCCTCACTGGCCGGCAGTGAAGCCATCCCCGCAGCCCGCTGCGGTGCAGGTAGACG1320              AGGAGGACAGCTCCGAATCCGAGGGCACCGTGGGCCCGCTCCTGAAGGGCCAACCTCGGG1380              CACTGGGAGGCACGGCGGCCGGAGGAGGAGCTGCCCCCGTCGCGTCTGGAGCGGCCGCAG1440              GAGGCGTCGCCCTTGTCCCCAAGGAAGATTCTCGCTTCTCGGCGCCCAGGGTCTCCTTGG1500              CGGAGCAGGACGCGCCGGTGGCGCCTGGGCGCTCCCCGCTGGCCACCTCGGTGGTGGATT1560              TCATCCACGTGCCCATCCTGCCTCTCAACCACGCTTTCCTGGCCACCCGCACCAGGCAGC1620              TGCTGGAGGGGGAGAGCTACGACGGCGGGGCCGCGGCCGCCAGCCCCTTCGTCCCGCAGC1680              GGGGCTCCCCCTCTGCCTCGTCCACCCCTGTGGCGGGCGGCGACTTCCCCGACTGCACCT1740              ACCCGCCCGACGCCGAGCCCAAAGATGACGCGTTCCCCCTCTACGGCGACTTCCAGCCGC1800              CCGCCCTCAAGATAAAGGAGGAGGAAGAAGCCGCCGAGGCCGCGGCGCGCTCCCCGCGTA1860              CGTACCTGGTGGCTGGTGCAAACCCCGCCGCCTTCCCGGACTTCCAGCTGGCAGCGCCGC1920              CGCCACCCTCGCTGCCGCCTCGAGTGCCCTCGTCCAGACCCGGGGAAGCGGCGGTGGCGG1980              CCTCCCCAGGCAGTGCCTCCGTCTCCTCCTCGTCCTCGTCGGGGTCGACCCTGGAGTGCA2040              TCCTGTACAAGGCAGAAGGCGCGCCGCCCCAGCAGGGCCCCTTCGCGCCGCTGCCCTGCA2100              AGCCTCCGGGCGCCGGCGCCTGCCTGCTCCCGCGGGACGGCCTGCCCTCCACCTCCGCCT2160              CGGGCGCAGCCGCCGGGGCCGCCCCTGCGCTCTACCCGACGCTCGGCCTCAACGGACTCC2220              CGCAACTCGGCTACCAGGCCGCCGTGCTCAAGGAGGGCCTGCCGCAGGTCTACACGCCCT2280              ATCTCAACTACCTGAGGCCGGATTCAGAAGCCAGTCAGAGCCCACAGTACAGCTTCGAGT2340              CACTACCTCAGAAGATTTGTTTGATCTGTGGGGATGAAGCATCAGGCTGTCATTATGGTG2400              TCCTCACCTGTGGGAGCTGTAAGGTCTTCTTTAAAAGGGCAATGGAAGGGCAGCATAACT2460              ATTTATGTGCTGGAAGAAATGACTGCATTGTTGATAAAATCCGCAGGAAAAACTGCCCGG2520              CGTGTCGCCTTAGAAAGTGCTGTCAAGCTGGCATGGTCCTTGGAGGGCGAAAGTTTAAAA2580              AGTTCAATAAAGTCAGAGTCATGAGAGCACTCGATGCTGTTGCTCTCCCACAGCCAGTGG2640              GCATTCCAAATGAAAGCCAACGAATCACTTTTTCTCCAAGTCAAGAGATACAGTTAATTC2700              CCCCTCTAATCAACCTGTTAATGAGCATTGAACCAGATGTGATCTATGCAGGACATGACA2760              ACACAAAGCCTGATACCTCCAGTTCTTTGCTGACGAGTCTTAATCAACTAGGCGAGCGGC2820              AACTTCTTTCAGTGGTAAAATGGTCCAAATCTCTTCCAGGTTTTCGAAACTTACATATTG2880              ATGACCAGATAACTCTCATCCAGTATTCTTGGATGAGTTTAATGGTATTTGGACTAGGAT2940              GGAGATCCTACAAACATGTCAGTGGGCAGATGCTGTATTTTGCACCTGATCTAATATTAA3000              ATGAACAGCGGATGAAAGAATCATCATTCTATTCACTATGCCTTACCATGTGGCAGATAC3060              CGCAGGAGTTTGTCAAGCTTCAAGTTAGCCAAGAAGAGTTCCTCTGCATGAAAGTATTAC3120              TACTTCTTAATACAATTCCTTTGGAAGGACTAAGAAGTCAAAGCCAGTTTGAAGAGATGA3180              GATCAAGCTACATTAGAGAGCTCATCAAGGCAATTGGTTTGAGGCAAAAAGGAGTTGTTT3240              CCAGCTCACAGCGTTTCTATCAGCTCACAAAACTTCTTGATAACTTGCATGATCTTGTCA3300              AACAACTTCACCTGTACTGCCTGAATACATTTATCCAGTCCCGGGCGCTGAGTGTTGAAT3360              TTCCAGAAATGATGTCTGAAGTTATTGCTGCACAGTTACCCAAGATATTGGCAGGGATGG3420              TGAAACCACTTCTCTTTCATAAAAAGTGAATGTCAATTATTTTTCAAAGAATTAAGTGTT3480              GTGGTATGTCTTTCGTTTTGGTCAGGATTATGACGTCTCGAGTTTTTATAATATTCTGAA3540              AGGGAATTCCTGCAGCCCGGGGGATCCACTAGTTCTAGAGGATCCAGACATGATAAGATA3600              CATTGATGAGTTTGGACAAACCACAACTAGAATGCAGTGAAAAAAATGCTTTATTTGTGA3660              AATTTGTGATGCTATTGCTTTATTTGTAACCATTATAAGCTGCAATAAACAAGTTAACAA3720              CAACAATTGCATTCATTTTATGTTTCAGGTTCAGGGGGAGGTGTGGGAGGTTTTTTAAAG3780              CAAGTAAAACCTCTACAAATGTGGTATGGCTGATTATGATCCTGCAAGCCTCGTCGTCTG3840              GCCGGACCACGCTATCTGTGCAAGGTCCCCGGACGCGCGCTCCATGAGCAGAGCGCCCGC3900              CGCCGAGGCAAGACTCGGGCGGCGCCCTGCCCGTCCCACCAGGTCAACAGGCGGTAACCG3960              GCCTCTTCATCGGGAATGCGCGCGACCTTCAGCATCGCCGGCATGTCCCCTGGCGGACGG4020              GAAGTATCAGCTCGACCAAGCTTGGCGAGATTTTCAGGAGCTAAGGAAGCTAAAATGGAG4080              AAAAAAATCACTGGATATACCACCGTTGATATATCCCAATGGCATCGTAAAGAACATTTT4140              GAGGCATTTCAGTCAGTTGCTCAATGTACCTATAACCAGACCGTTCAGCTGCATTAATGA4200              ATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTTCCGCTTCCTCGCTC4260              ACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCG4320              GTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGC4380              CAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGC4440              CCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGA4500              CTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACC4560              CTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCAA4620              TGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTG4680              CACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCC4740              AACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGA4800              GCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACT4860              AGAAGGACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTT4920              GGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAG4980              CAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGG5040              TCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAA5100              AGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATA5160              TATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCG5220              ATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATA5280              CGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCG5340              GCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCT5400              GCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGT5460              TCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGC5520              TCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGA5580              TCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGT5640              AAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTC5700              ATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAA5760              TAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCA5820              CATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCA5880              AGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCT5940              TCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCC6000              GCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAA6060              TATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATT6120              TAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTGACGTC6180              TAAGAAACCATTATTATCATGACATTAACCTATAAAAATAGGCGTATCACGAGGCCCTTT6240              CGTC6244                                                                      (2) INFORMATION FOR SEQ ID NO:9:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 4963 base pairs                                                   (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: circular                                                        (ii) MOLECULE TYPE: DNA (genomic)                                             (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Human cytomegalovirus                                           (vii) IMMEDIATE SOURCE:                                                       (B) CLONE: pUHD BGR4                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                                       CTCGAGTTTACCACTCCCTATCAGTGATAGAGAAAAGTGAAAGTCGAGTTTACCACTCCC60                TATCAGTGATAGAGAAAAGTGAAAGTCGAGTTTACCACTCCCTATCAGTGATAGAGAAAA120               GTGAAAGTCGAGTTTACCACTCCCTATCAGTGATAGAGAAAAGTGAAAGTCGAGTTTACC180               ACTCCCTATCAGTGATAGAGAAAAGTGAAAGTCGAGTTTACCACTCCCTATCAGTGATAG240               AGAAAAGTGAAAGTCGAGTTTACCACTCCCTATCAGTGATAGAGAAAAGTGAAAGTCGAG300               CTCGGTACCCGGGTCGAGTAGGCGTGTACGGTGGGAGGCCTATATAAGCAGAGCTCGTTT360               AGTGAACCGTCAGATCGCCTGGAGACGCCATCCACGCTGTTTTGACCTCCATAGAAGACA420               CCGGGACCGATCCAGCCTCCGCGGCCCCGAATTCCGGCCACGACCATGACCATGACCCTC480               CACACCAAAGCATCTGGGATGGCCCTACTGCATCAGATCCAAGGGAACGAGCTGGAGCCC540               CTGAACCGTCCGCAGCTCAAGATCCCCCTGGAGCGGCCCCTGGGCGAGGTGTACCTGGAC600               AGCAGCAAGCCCGCCGTGTACAACTACCCCGAGGGCGCCGCCTACGAGTTCAACGCCGCG660               GCCGCCGCCAACGCGCAGGTCTACGGTCAGACCGGCCTCCCCTACGGCCCCGGGTCTGAG720               GCTGCGGCGTTCGGCTCCAACGGCCTGGGGGGTTTCCCCCCACTCAACAGCGTGTCTCCG780               AGCCCGCTGATGCTACTGCACCCGCCGCCGCAGCTGTCGCCTTTCCTGCAGCCCCACGGC840               CAGCAGGTGCCCTACTACCTGGAGAACGAGCCCAGCGGCTACACGGTGCGCGAGGCCGGC900               CCGCCGGCATTCTACAGGCCAAATTCAGATAATCGACGCCAGGGTGGCAGAGAAAGATTG960               GCCAGTACCAATGACAAGGGAAGTATGGCTATGGAATCTGCCAAGGAGACTCGCTACTGT1020              GCAGTGTGCAATGACTATGCTTCAGGCTACCATTATGGAGTCTGGTCCTGTGAGGGCTGC1080              AAGGCCTTCTTCAAGAGAAGTATTCAAGGACATAACGACTATATGTGTCCAGCCACCAAC1140              CAGTGCACCATTGATAAAAACAGGAGGAAGAGCTGCCAGGCCTGCCGGCTCCGCAAATGC1200              TACGAAGTGGGAATGATGAAAGGTGGGATACGAAAAGACCGAAGAGGAGGGAGAATGTTG1260              AAACACAAGCGCCAGAGAGATGATGGGGAGGGCAGGGGTGAAGTGGGGTCTGCTGGAGAC1320              ATGAGAGCTGCCAACCTTTGGCCAAGCCCGCTCATGATCAAACGCTCTAAGAAGAACAGC1380              CTGGCCTTGTCCCTGACGGCCGACCAGATGGTCATGGCCTTGTTGGATGCTGAGCCCCCC1440              ATACTCTATTCCGAGTATGATCCTACCAGACCCTTCAGTGAAGCTTCGATGATGGGCTTA1500              CTGACCAACCTGGCAGACAGGGAGCTGGTTCACATGATCAACTGGGCGAAGAGGGTGCCA1560              GGCTTTGTGGATTTGACCCTCCATGATCAGGTCCACCTTCTAGAATGTGCCTGGCTAGAG1620              ATCCTGATGATTGGTCTCGTCTGGCGCTCCATGGAGCACCCAGTGAAGCTACTGTTTGCT1680              CCTAACTTGCTCTTGGACAGGAACCAGGGAAAATGTGTAGAGGGCATGGTGGAGATCTTC1740              GACATGCTGCTGGCTACATCATCTCGGTTCCGCATGATGAATCTGCAGGGAGAGGAGTTT1800              GTGTGCCTCAAATCTATTATTTTGCTTAATTCTGGAGTGTACACATTTCTGTCCAGCACC1860              CTGAAGTCTCTGGAAGAGAAGGACCATATCCACCGAGTCCTGGACAAGATCACAGACACT1920              TTGATCCACCTGATGGCCAAGGCAGGCCTGACCCTGCAGCAGCAGCACCAGCGGCTGGCC1980              CAGCTCCTCCTCATCCTCTCCCACATCAGGCACATGAGTAACAAAGGCATGGAGCATCTG2040              TACAGCATGAAGTGCAAGAACGTGGTGCCCCTCTATGACCTGCTGCTGGAGATGCTGGAC2100              GCCCACCGCCTACATGCGCCCACTAGCCGTGGAGGGGCATCCGTGGAGGAGACGGACCAA2160              AGCCACTTGGCCACTGCGGGCTCTACTTCATCGCATTCCTTGCAAAAGTATTACATCACG2220              GGGGAGGCAGAGGGTTTCCCTGCCACAGTCTGAGAGCTCCCTGGCGGAATTCGAGCTCGG2280              TACCCGGGGATCCTCTAGAGGATCCAGACATGATAAGATACATTGATGAGTTTGGACAAA2340              CCACAACTAGAATGCAGTGAAAAAAATGCTTTATTTGTGAAATTTGTGATGCTATTGCTT2400              TATTTGTAACCATTATAAGCTGCAATAAACAAGTTAACAACAACAATTGCATTCATTTTA2460              TGTTTCAGGTTCAGGGGGAGGTGTGGGAGGTTTTTTAAAGCAAGTAAAACCTCTACAAAT2520              GTGGTATGGCTGATTATGATCCTGCAAGCCTCGTCGTCTGGCCGGACCACGCTATCTGTG2580              CAAGGTCCCCGGACGCGCGCTCCATGAGCAGAGCGCCCGCCGCCGAGGCAAGACTCGGGC2640              GGCGCCCTGCCCGTCCCACCAGGTCAACAGGCGGTAACCGGCCTCTTCATCGGGAATGCG2700              CGCGACCTTCAGCATCGCCGGCATGTCCCCTGGCGGACGGGAAGTATCAGCTCGACCAAG2760              CTTGGCGAGATTTTCAGGAGCTAAGGAAGCTAAAATGGAGAAAAAAATCACTGGATATAC2820              CACCGTTGATATATCCCAATGGCATCGTAAAGAACATTTTGAGGCATTTCAGTCAGTTGC2880              TCAATGTACCTATAACCAGACCGTTCAGCTGCATTAATGAATCGGCCAACGCGCGGGGAG2940              AGGCGGTTTGCGTATTGGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGT3000              CGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGA3060              ATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCG3120              TAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAA3180              AAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTT3240              TCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCT3300              GTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCAATGCTCACGCTGTAGGTATCT3360              CAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCC3420              CGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTT3480              ATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGC3540              TACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGGACAGTATTTGGTAT3600              CTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAA3660              ACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAA3720              AAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGA3780              AAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCT3840              TTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGA3900              CAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATC3960              CATAGTTGCCTGATCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGC4020              CCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATA4080              AACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATC4140              CAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGC4200              AACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCA4260              TTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAA4320              GCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCA4380              CTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTT4440              TCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGT4500              TGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTG4560              CTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGA4620              TCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACC4680              AGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCG4740              ACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAG4800              GGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGG4860              GTTCCGCGCACATTTCCCCGAAAAGTGCCACCTGACGTCTAAGAAACCATTATTATCATG4920              ACATTAACCTATAAAAATAGGCGTATCACGAGGCCCTTTCGTC4963                               (2) INFORMATION FOR SEQ ID NO:10:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 42 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                                      TCGAGTTTACCACTCCCTATCAGTGATAGAGAAAAGTGAAAG42                                  __________________________________________________________________________

We claim:
 1. An isolated DNA molecule for integrating a polynucleotidesequence encoding a tetracycline-controllable transactivator (tTA) at apredetermined location in a second target DNA molecule, the tTAcomprising a prokaryotic Tet repressor operably linked to a polypeptidewhich directly or indirectly activates transcription in eucaryoticcells, the DNA molecule comprising a polynucleotide sequence encodingthe tTA flanked at 5' and 3' ends by additional polynucleotide sequencesof sufficient length for homologous recombination between the DNAmolecule and the second target DNA molecule at a predetermined location.2. The DNA molecule of claim 1, wherein the additional polynucleotidesequences flanking the polynucleotide sequence encoding the tTA are of agene of interest, or regulatory region thereof, into which the DNAmolecule is inserted.
 3. The DNA molecule of claim 2, wherein, uponintegration of the DNA molecule into the gene of interest, or regulatoryregion thereof, expression of the tTA is controlled by regulatoryelements of the gene of interest.
 4. The DNA molecule of claim 1,wherein the Tet repressor of the tTA is a Tn10-derived Tet repressor. 5.The DNA molecule of claim 1, wherein the polypeptide of the tTA whichdirectly or indirectly activates transcription in eucaryotic cells isfrom herpes simplex virus virion protein
 16. 6. The DNA molecule ofclaim 1, wherein the polypeptide of the tTA which directly or indirectlyactivates transcription in eucaryotic cells is selected from the groupconsisting of acidic, proline-rich, serine/threonine-rich andglutamine-rich transcriptional activation polypeptides.
 7. The DNAmolecule of claim 1, wherein the polypeptide of the tTA which directlyor indirectly activates transcription in eucaryotic cells is aninteraction domain selected from the group consisting of a leucinezipper domain, a helix-loop-helix domain and a zinc finger domain. 8.The DNA molecule of claim 1, wherein the polypeptide of the tTA whichdirectly or indirectly activates transcription in eucaryotic cells is aninteraction domain from a TATA binding protein.
 9. The DNA molecule ofclaim 1, further comprising a polynucleotide sequence encoding aselectable marker.
 10. The DNA molecule of claim 9, wherein thenucleotide sequence encoding the selectable marker is a tk gene or aneomycin resistance gene.
 11. An isolated DNA molecule for integrating apolynucleotide sequence encoding a tetracycline-controllabletransactivator (tTA) and a tTA-responsive promoter within apredetermined gene of interest in a second target DNA molecule, the DNAmolecule comprising:a) a first polynucleotide sequence comprising a 5'flanking regulatory region of the gene of interest, operably linked to:b) a second polynucleotide sequence encoding a tTA, the tTA comprising aprokaryotic Tet repressor operably linked to a polypeptide whichdirectly or indirectly activates transcription in eucaryotic cells; andc) a third polynucleotide sequence comprising a tTA-responsive promoter,operably linked to: d) a fourth polynucleotide sequence comprising atleast a portion of a coding region of the gene of interest;wherein thefirst and fourth polynucleotide sequences are of sufficient length forhomologous recombination between the DNA molecule and the gene ofinterest in the second target DNA molecule such that expression of thetTA is controlled by 5' regulatory elements of the gene of interest andexpression of the gene of interest is controlled by the tTA-responsivepromoter.
 12. The DNA molecule of claim 11, further comprising a fifthpolynucleotide sequence encoding a selectable marker operably linked toa regulatory sequence, wherein the fifth polynucleotide sequence islocated between the second and third polynucleotide sequences.
 13. TheDNA molecule of claim 12, wherein the fifth polynucleotide sequenceencoding the selectable marker is a tk gene or a neomycin resistancegene.
 14. The DNA molecule of claim 11, further comprising a fifthpolynucleotide sequence comprising a transcriptional terminator signal,a transcriptional insulator or a matrix attachment region, wherein thefifth polynucleotide sequence is located between the second and thirdpolynucleotide sequences.
 15. The DNA molecule of claim 12, furthercomprising a sixth polynucleotide sequence comprising a transcriptionalterminator signal, a transcriptional insulator or a matrix attachmentregion, wherein the sixth polynucleotide sequence is located between thefifth and third polynucleotide sequences.
 16. The DNA molecule of claim11, wherein the Tet repressor of the tTA is a Tn10-derived Tetrepressor.
 17. The DNA molecule of claim 11, wherein the polypeptide ofthe tTA which directly or indirectly activates transcription ineucaryotic cells is from herpes simplex virus virion protein
 16. 18. TheDNA molecule of claim 11, wherein the polypeptide of the tTA whichdirectly or indirectly activates transcription in eucaryotic cells isselected from the group consisting of acidic, proline-rich,serine/threonine-rich and glutamine-rich transcriptional activationpolypeptides.
 19. The DNA molecule of claim 11, wherein the polypeptideof the tTA which directly or indirectly activates transcription ineucaryotic cells is an interaction domain selected from the groupconsisting of a leucine zipper domain, a helix-loop-helix domain and azinc finger domain.
 20. The DNA molecule of claim 11, wherein thepolypeptide of the tTA which directly or indirectly activatestranscription in eucaryotic cells is an interaction domain from a TATAbinding protein.
 21. The DNA molecule of claim 11, wherein thetTA-responsive promoter of the third nucleotide sequence comprises aminimal promoter operably linked to at least one tet operator sequence.22. The DNA molecule of claim 21, wherein the minimal promoter isderived from a cytomegalovirus immediate early gene promoter or a herpessimplex virus thymidine kinase gene promoter.
 23. The DNA molecule ofclaim 11, wherein the gene of interest is a human gene.
 24. The DNAmolecule of claim 23, wherein the human gene encodes a gene productselected from the group consisting of adenosine deaminase, Factor VIII,Factor IX, dystrophin, β-globin, LDL receptor, CFTR, insulin,erythropoietin, anti-angiogenesis factors, growth hormone,glucocerebrosidase, β-glucouronidase, α1-antitrypsin, phenylalaninehydroxylase, tyrosine hydroxylase, ornithine transcarbamylase,arginosuccinate synthetase, UDP-glucuronysyl transferase, apoA1, MDR1,MRP, TNF, soluble TNF receptor, an interleukins, an interferon, acytokine, a growth factor and a tumor suppressor gene.
 25. A eucaryotichost cell comprising the DNA molecule of claim 1, wherein the DNAmolecule is integrated at a predetermined location in a second targetDNA molecule in the host cell.
 26. The host cell of claim 25, furthercomprising a gene of interest operably linked to a tTA-responsivetranscriptional promoter.
 27. The host cell of claim 26, wherein thetTA-responsive promoter comprises a minimal promoter operably linked toat least one tet operator sequence.
 28. The host cell of claim 27,wherein the minimal promoter is derived from a cytomegalovirus immediateearly gene promoter or a herpes simplex virus thymidine kinase genepromoter.
 29. The host cell of claim 25, which is a mammalian cell. 30.The host cell of claim 29, which is a human cell.
 31. The host cell ofclaim 29, which is a mouse embryonic stem cell.
 32. The host cell ofclaim 25, which is a yeast cell or a fungal cell.
 33. The host cell ofclaim 25, wherein the cell is an insect cell and the second target DNAmolecule is an insect gene or a baculovirus gene.
 34. A method forinhibiting transcription of the gene of interest operatively linked tothe tTA-responsive promoter in the host cell of claim 26, comprisingcontacting the cell with tetracycline or a tetracycline analogue.
 35. Ahost cell comprising the nucleic acid of claim 11, wherein the nucleicacid is integrated into the predetermined gene of interest in a secondtarget DNA molecule in the host cell.
 36. The host cell of claim 35,which is a mammalian cell.
 37. The host cell of claim 36, which is ahuman cell.
 38. The host cell of claim 36, which is a mouse embryonicstem cell.
 39. The host cell of claim 35, which is a yeast cell or afungal cell.
 40. The host cell of claim 35, wherein the cell is aninsect cell and the gene of interest is an insect gene or a baculovirusgene.
 41. A method for inhibiting transcription of the eucaryotic geneof interest in the host cell of claim 35, comprising contacting the cellwith tetracycline or a tetracycline analogue.
 42. A process forproducing and isolating a gene product encoded by the gene of interestoperably linked to the tTA-responsive transcriptional promoter in thecell of claim 26, comprising:a) growing cells in a culture medium in thepresence of tetracycline or a tetracycline analogue; b) reducing theconcentration of tetracycline or the tetracycline analogue to stimulatetranscription of the gene of interest; c) further culturing the cellsuntil a desired amount of the gene product encoded by the gene ofinterest is produced by the cells; and b) isolating the gene productfrom harvested cells or from the culture medium.
 43. The process ofclaim 42, wherein the cells are mammalian cells.
 44. The process ofclaim 42, wherein the cells are yeast or fungal cells.
 45. A method forproducing a host cell having a DNA molecule encoding atetracycline-controllable transactivator (tTA) integrated at apredetermined location in a second target DNA molecule within the cell,comprising:a) introducing the DNA molecule of claim 1 into a populationof cells under conditions suitable for homologous recombination betweenthe DNA encoding the tTA and the second target DNA molecule; and b)selecting a cell in which the DNA encoding the tTA has integrated at apredetermined location within the second target DNA molecule.